Multivalent and multispecific DR5-binding fusion proteins

ABSTRACT

The disclosure relates generally to molecules that specifically engage death receptor 5 (DR5), a member of the TNF receptor superfamily (TNFRSF). More specifically the disclosure relates to multivalent and multispecific molecules that bind at least DR5.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/193,309, filed Jul. 16, 2015, the contents of which are incorporatedherein by reference in their entirety.

The contents of the text file named “INHI021001US_ST25.txt”, which wascreated on Oct. 4, 2016 and is 210 KB in size, are hereby incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The disclosure relates generally to molecules that specifically engagedeath receptor 5 (DR5), a member of the TNF receptor superfamily(TNFRSF). More specifically the disclosure relates to multivalent andmultispecific molecules that bind at least DR5.

BACKGROUND OF THE INVENTION

The tumor necrosis factor receptor superfamily consists of severalstructurally related cell surface receptors. Activation by multimericligands is common feature of many of these receptors. Many members ofthe TNFRSF have therapeutic utility in numerous pathologies, ifactivated properly. Importantly to properly agonize this receptor familyoften requires higher order clustering and conventional bivalentantibodies are not ideal for this. Therefore, there exists a therapeuticneed for more potent agonist molecules of the TNFRSF.

SUMMARY OF THE INVENTION

The disclosure provides multivalent fusion polypeptides that bind atleast death receptor 5 (DR5, also known as TRAIL receptor 2 (TRAILR2),or tumor necrosis factor receptor superfamily member 10B (TNFRSF10B)).These DR5 binding fusion polypeptides are also referred to herein asDR5-targeting molecules. DR5 is a member of the TNF receptor superfamily(TNFRSF) and a cell surface receptor of the TNF-receptor superfamilythat binds TNF-related apoptosis-inducing ligand (TRAIL). TRAIL evolvedto play critical roles in mammalian development and host defense byselectively eradicating unwanted, infected and malignant cells fromhealthy cell populations. On binding the TNF receptor family members DR4or DR5, TRAIL induces cell death via caspase-dependent apoptosis. DR5appears to be the primary receptor on tumor cells that facilitates theobserved tumor biased activity of the TRAIL pathway. DR5 is activated bythe natural ligand TRAIL, which brings three DR5 receptors within closeproximity thereby activating intracellular caspase-8 and initiatingactivation of other death-inducing caspases, such as caspases-9 andcaspases-3. Thus initiation of this cell death pathway requiresclustering of DR5 receptors for efficient cell death.

Conventional antibodies targeting members of the TNF receptorsuperfamily (TNFRSF) have been shown to require an exogenouscrosslinking to achieve sufficient agonist activity, as evidenced by thenecessity for Fc-gamma Receptor (FcγRs) for the activity antibodies toDR4, DR5, GITR and OX40 (Ichikawa et at 2001 al Nat. Med. 7, 954-960, Liet at 2008 Drug Dev. Res. 69, 69-82; Pukac et al 2005 Br. J. Cancer 92,1430-1441; Yanda et at 2008 Ann. Oncol. 19, 1060-1067; Yang et al 2007Cancer Lett. 251:146-157; Bulliard et al 2013 JEM 210(9): 1685; Bulliardet at 2014 Immunol and Cell Biol 92: 475-480). In addition tocrosslinking via FcγRs, other exogenous agents including addition of theoligomeric ligand or antibody binding entities (e.g. protein A andsecondary antibodies) have be demonstrated to enhance anti-TNFRSFantibody clustering and downstream signaling. For instance, in vitroagonist activity of the CD137 antibody, PF-05082566, requirescrosslinking via a secondary antibody (Fisher et at Cancer ImmunolImmunother 2012 61:1721-1733). These findings suggest the need forclustering of TNFRSFs beyond a dimer.

Efforts to clinically exploit the TRAIL pathway for cancer therapyrelied upon a recombinant version of the natural ligand TRAIL andantibodies specific for DR5. Antibody agonists targeting DR5 required acrosslinking agent in preclinical in vitro experiments. For example, theaddition of the DR5 ligand TRAIL enhanced the apoptosis inducing abilityof an anti-DR5 antibody, AMG655 (Graves et al 2014 Cancer Cell 26:177-189). Conventional antibodies are bivalent and capable clusteringonly two DR5 receptors (one per each FAB arm). Consistent with othermembers of the TNFRSF, clustering of two DR5 receptors is insufficientto mediate signaling and activate the cell death pathway in vitro.Surprisingly in vivo administration of DR5 targeting antibodies inpre-clinical mouse models of human cancers showed significant activityin a wide variety of tumor types. This activity was later shown to bedependent on mouse FcgammaR (FcγR) receptors. Clinical studies in humansfailed to reproduce the robust responses seen in these pre-clinicalmouse models. The lack of activity in humans is hypothesized to be dueto insufficient antibody crosslinking. This may be due to differences inserum IgG, FcγR and or TRAIL concentrations between immune compromisedmice and human cancer patients.

The present disclosure provides multivalent fusion proteins targetingDR5 that are capable of potently agonizing DR5 signaling mediatingdirect cell death. The fusion proteins of the present disclosure can bebivalent, trivalent, tetravalent, pentavalent, or hexavalent.Importantly, the fusion proteins of the present disclosure are capableof eliciting apoptosis of DR5 expressing cells independently ofexogenous crosslinking agents.

In some embodiments, the fusion proteins of the present disclosureincorporate a binding domain (DR5BD) that binds DR5. In preferredembodiments, the DR5 binding DR5BD does not bind DR4, decoy R1, decoyR2, Osteopontin, or any other TNFRSF member. In preferred embodimentsthe DR5 binding DR5BD binds human and cynomolgus monkey DR5. In someembodiments, the DR5 binding DR5BD blocks the interaction of DR5 and itsligand TRAIL. In other embodiments, the DR5 binding DR5BD does not blockthe interaction of DR5 and its ligand TRAIL. In some embodiments, thefusion protein of the present disclosure incorporates multiple DR5binding DR5BDs that recognize distinct epitopes on DR5. In someembodiments, the fusion protein of the present disclosure incorporatesmultiple DR5 binding DR5BDs, wherein some DR5BDs block the DR5-TRAILinteraction and other do not block the DR5-TRAIL interaction. Inpreferred embodiments, DR5 targeting fusion proteins of the presentdisclosure induce direct cell death of tumor cells. The DR5 targetingfusion proteins of the present disclosure have utility in treatingtumors both hematologic and solid in nature.

The present disclosure provides multivalent DR5 binding fusion proteins,which comprise 2 or more DR5 binding domains (DR5BDs). In someembodiments, the fusion proteins of the present disclosure have utilityin treating neoplasms. In some embodiments, the fusion proteins of thepresent disclosure bind DR5 expressed on a tumor cell. In someembodiments, the fusion protein contains two or more different DR5BDs,where each DR5BD binds DR5. In some embodiments, the fusion proteincontains multiple copies of a DR5BD that binds DR5. For example, in someembodiments, the fusion protein contains at least two copies of a DR5BDthat binds DR5. In some embodiments, the fusion protein contains atleast three copies of a DR5BD that binds DR5. In some embodiments, thefusion protein contains at least four copies of a DR5BD that binds DR5.In some embodiments, the fusion protein contains at least five copies ofa DR5BD that binds DR5. In some embodiments, the fusion protein containsat least six copies of a DR5BD that binds DR5. In some embodiments, thefusion protein contains six or more copies of a DR5BD that binds DR5.

Multivalent DR5 binding fusion proteins of the present disclosure arecapable of inducing direct cell death of damaged, transformed, virallyinfected, or neoplastic cells without the need for exogenouscrosslinking agents. In addition, DR5 binding fusion proteins of thepresent disclosure do not induce direct cell death of normal,non-transformed cells, non-virally infected or non-neoplastic cells.Importantly, the DR5BDs and fusion proteins composed thereof of thepresent disclosure have reduced or eliminated recognition bypre-existing antibodies directed toward single domain antibodies presentin some human subjects.

TAS266 is a tetravalent humanized DR5-targeting nanobody-basedtherapeutic, which displays superior apoptosis inducing capacitycompared to bivalent antibodies, without the need for additionalcrosslinking by FcγRs. (Huet, H. A., et al., Multivalent nanobodiestargeting death receptor 5 elicit superior tumor cell killing throughefficient caspase induction. mAbs Vol. 6, Iss. 6, 2014).

It has previously been predicted that approximately half of healthyhuman subjects have pre-existing antibodies recognizing human singledomain antibodies, known as human anti-VH autoantibodies (HAVH), whichtarget an epitope within human VH domains (Holland et al. J Clin Immunol(2013) 33:1192-1203)). Thus, it expected that humanized camelid-derivedVHHs would also be recognized by HAVH autoantibodies as the targetepitope seems to be cryptic and located within human germline frameworkregions. The interaction of HAVH autoantibodies (also called anti-drugantibodies (ADA) or anti-single domain antibodies (ASDA), herein) cancause enhanced clustering and activation. In agreement with thishypothesis, in a Phase I clinical trial, administration of TAS266induced elevated AST and ALT levels indicative of hepatotoxicity.Elevated enzyme levels occurred in 3 out of 4 patients leading totermination of the TAS266 trial. It was noted that the 3 patientsexhibiting clinical signs of hepatotoxicity had pre-existing ADA leadingtrial investigators to suspect that ADA-induced hyper-clustering of theDR5 receptor causing toxicity. It was noted that the one patient withoutADA had no signs of toxicity (Isaacs R, Bilic S, Kentsch K, Huet H A,Hofmann M, Rasco D, Kundamal N, Tang Z, Cooksey J, Mahipal A. Unexpectedhepatotoxicity in a phase I study of TAS266, a novel tetravalentagonistic Nanobody® targeting the DR5 receptor. Papadopoulos KP1, CancerChemother Pharmacol. 2015 May; 75(5):887-95. doi:10.1007/s00280-015-2712-0. Epub 2015 Feb. 27.). In support of this idea,it has been well-documented that aggregated forms of DR5 agonists inducehepatotoxicity whereas non-aggregated forms do not (J Lemke, S vonKarstedt, J Zinngrebe and H Walczak. Getting TRAIL back on track forcancer therapy. Cell Death and Differentiation (2014) 21, 1350-1364).

In some embodiments, the fusion protein contains at least one DR5BD thatcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 15-91. In some embodiments, the fusion protein contains twoor more copies of a DR5BD that comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 15-91. In some embodiments, thefusion protein contains three or more copies of a DR5BD that comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:15-91. In some embodiments, the fusion protein contains four or morecopies of a DR5BD that comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO: 15-91. In some embodiments, thefusion protein contains five or more copies of a DR5BD that comprises anamino acid sequence selected from the group consisting of SEQ ID NO:15-91. In some embodiments, the fusion protein contains six or morecopies of a DR5BD that comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO: 15-91.

In some embodiments, the fusion protein contains at least one DR5BD thatcomprises a complementarity determining region 1 (CDR1) comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 31,128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176, 178, 181, and188; a complementarity determining region 2 (CDR2) comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 28, 129,131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174,177, 179, 182, 184, 185, and 189; and a complementarity determiningregion 3 (CDR3) comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 130, 136, 140, 144-158, 161, 165, 170,175, 180, 183, 186, 187, and 190. In some embodiments, the fusionprotein contains two or more copies of a DR5BD that comprises a CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176,178, 181, and 188; a CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 28, 129, 131-133, 135, 137, 139,143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185,and 189; and a CDR3 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 130, 136, 140, 144-158, 161, 165, 170,175, 180, 183, 186, 187, and 190. In some embodiments, the fusionprotein contains three or more copies of a DR5BD that comprises a CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176,178, 181, and 188; a CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 28, 129, 131-133, 135, 137, 139,143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185,and 189; and a CDR3 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 130, 136, 140, 144-158, 161, 165, 170,175, 180, 183, 186, 187, and 190. In some embodiments, the fusionprotein contains four or more copies of a DR5BD that comprises a CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176,178, 181, and 188; a CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 28, 129, 131-133, 135, 137, 139,143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185,and 189; and a CDR3 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 130, 136, 140, 144-158, 161, 165, 170,175, 180, 183, 186, 187, and 190. In some embodiments, the fusionprotein contains five or more copies of a DR5BD that comprises a CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176,178, 181, and 188; a CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 28, 129, 131-133, 135, 137, 139,143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185,and 189; and a CDR3 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 130, 136, 140, 144-158, 161, 165, 170,175, 180, 183, 186, 187, and 190. In some embodiments, the fusionprotein contains six or more copies of a DR5BD that comprises a CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 31, 128, 134, 138, 141, 142, 159, 162, 163, 168, 173, 176,178, 181, and 188; a CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 28, 129, 131-133, 135, 137, 139,143, 160, 164, 166, 167, 169, 171, 172, 174, 177, 179, 182, 184, 185,and 189; and a CDR3 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 130, 136, 140, 144-158, 161, 165, 170,175, 180, 183, 186, 187, and 190.

In some embodiments, the fusion protein contains at least one DR5BD thatcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 15-91 and at least one immunoglobulin Fc region polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1-5 or 127. In some embodiments, the fusion protein containstwo or more copies of a DR5BD that comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 15-91 and at least oneimmunoglobulin Fc region polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-5 or 127. In someembodiments, the fusion protein contains three or more copies of a DR5BDthat comprises an amino acid sequence selected from the group consistingof SEQ ID NO: 15-91 and at least one immunoglobulin Fc regionpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-5 or 127. In some embodiments, the fusionprotein contains four or more copies of a DR5BD that comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 15-91 andat least one immunoglobulin Fc region polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1-5 or127. In some embodiments, the fusion protein contains five or morecopies of a DR5BD that comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO: 15-91 and at least one immunoglobulinFc region polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1-5 or 127. In some embodiments, thefusion protein contains six or more copies of a DR5BD that comprises anamino acid sequence selected from the group consisting of SEQ ID NO:15-91 and at least one immunoglobulin Fc region polypeptide comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs:1-5 or 127.

In some embodiments, the fusion protein contains at least one DR5BD thatcomprises a CDR1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 31, 128, 134, 138, 141, 142, 159, 162,163, 168, 173, 176, 178, 181, and 188; a CDR2 comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 28, 129,131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172, 174,177, 179, 182, 184, 185, and 189; and a CDR3 comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 130, 136, 140,144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and at leastone immunoglobulin Fc region polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-5 or 127.In some embodiments, the fusion protein contains two or more copies of aDR5BD that comprises a CDR1 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 31, 128, 134, 138, 141, 142,159, 162, 163, 168, 173, 176, 178, 181, and 188; a CDR2 comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 28,129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172,174, 177, 179, 182, 184, 185, and 189; and a CDR3 comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 130, 136,140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and atleast one immunoglobulin Fc region polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-5 or 127.In some embodiments, the fusion protein contains three or more copies ofa DR5BD that comprises a CDR1 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 31, 128, 134, 138, 141, 142,159, 162, 163, 168, 173, 176, 178, 181, and 188; a CDR2 comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 28,129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172,174, 177, 179, 182, 184, 185, and 189; and a CDR3 comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 130, 136,140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and atleast one immunoglobulin Fc region polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-5 or 127.In some embodiments, the fusion protein contains four or more copies ofa DR5BD that comprises a CDR1 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 31, 128, 134, 138, 141, 142,159, 162, 163, 168, 173, 176, 178, 181, and 188; a CDR2 comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 28,129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172,174, 177, 179, 182, 184, 185, and 189; and a CDR3 comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 130, 136,140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and atleast one immunoglobulin Fc region polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-5 or 127.In some embodiments, the fusion protein contains five or more copies ofa DR5BD that comprises a CDR1 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 31, 128, 134, 138, 141, 142,159, 162, 163, 168, 173, 176, 178, 181, and 188; a CDR2 comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 28,129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172,174, 177, 179, 182, 184, 185, and 189; and a CDR3 comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 130, 136,140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and atleast one immunoglobulin Fc region polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-5 or 127.In some embodiments, the fusion protein contains six or more copies of aDR5BD that comprises a CDR1 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 31, 128, 134, 138, 141, 142,159, 162, 163, 168, 173, 176, 178, 181, and 188; a CDR2 comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 28,129, 131-133, 135, 137, 139, 143, 160, 164, 166, 167, 169, 171, 172,174, 177, 179, 182, 184, 185, and 189; and a CDR3 comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 130, 136,140, 144-158, 161, 165, 170, 175, 180, 183, 186, 187, and 190; and atleast one immunoglobulin Fc region polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-5 or 127.

In some embodiments, the fusion protein comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 92-124. In someembodiments, the fusion protein comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 92-118. In someembodiments, the fusion protein comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 119-124.

The fusion proteins of the present disclosure are capable of enhancedclustering of TNFRSF members compared to non-cross-linked bivalentantibodies. The enhanced clustered of TNFRSF members mediated by thefusion proteins of the present disclosure induce enhancedTNFRSF-dependent signaling compared to non-cross-linked bivalentantibodies. In most embodiments, the fusion protein will incorporatemore than 2 DR5BDs, for example, three, four, five, or six. In someembodiments the fusion protein will incorporate DR5BDs and a bindingdomain directed toward non-TNFRSF member antigen. In these embodiments,the interaction of the non-TNFRSF antigen is capable of providing theadditional crosslinking function and TNFRSF activation is achieved withonly one or two DR5BDs. In these embodiments, the fusion protein ismultispecific, binding two distinct antigens. In other embodiments, thefusion protein incorporates three or more DR5BDs and a binding domaindirected toward an antigen other than DR5, wherein the interaction withthis additional antigen dose not enhance DR5 clustering beyond what isachieved by the DR5BD containing portion alone, but rather provides abiodistribution advantage, focusing the DR5 agonistic activity of thefusion protein to a specific site within a subject. For example, atetravalent DR5 binding fusion protein of the present disclosure mayinclude an additional antigen binding domain that focuses activity to aspecific site, yet does not enhance the agonistic activity beyond thatachieved by a tetravalent DR5 binding fusion protein lacking thisadditional antigen binding domain.

In some embodiments, DR5BDs of the present disclosure are derived fromantibodies or antibody fragments including scFv, Fabs, single domainantibodies (sdAb), V_(NAR), or VHHs. In preferred embodiments the DR5BDsare human or humanized sdAb. The sdAb fragments, can be derived fromVHH, V_(NAR), engineered VH or VK domains. VHHs can be generated fromcamelid heavy chain only antibodies. V_(NAR)s can be generated fromcartilaginous fish heavy chain only antibodies. Various methods havebeen implemented to generate monomeric sdAbs from conventionallyheterodimeric VH and VK domains, including interface engineering andselection of specific germline families. In other embodiments, theDR5BDs are derived from non-antibody scaffold proteins for example butnot limited to designed ankyrin repeat proteins (darpins), avimer,anticalin/lipocalins, centyrins and fynomers.

Generally the fusion proteins of the present disclosure consist of atleast two or more DR5BDs operably linked via a linker polypeptide. Theutilization of sdAb fragments as the specific DR5BD within the fusionthe present disclosure has the benefit of avoiding the heavy chain:lightchain mis-pairing problem common to many bi/multispecific antibodyapproaches. In addition, the fusion proteins of the present disclosureavoid the use of long linkers necessitated by many bispecificantibodies.

In some embodiments, all of the DR5BDs of the fusion protein recognizethe same epitope on DR5. For example, the fusion proteins of presentdisclosure may incorporate 2, 3, 4, 5, or 6 DR5BDs with distinctrecognition specificities toward various epitopes on DR5. In theseembodiments, the fusion proteins of the present disclosure with containmultiple DR5BDs that target distinct regions of DR5. In someembodiments, the DR5BDs may recognize different epitopes on DR5 orrecognize epitopes on DR5 and a distinct antigen. For example, thepresent disclosure provides multispecific fusion proteins incorporatingDR5BDs that bind DR5 and at least a second antigen.

In some embodiments, the fusion protein of the present disclosure iscomposed of a single polypeptide. In other embodiments, the fusionprotein of the present disclosure is composed of more than onepolypeptide. For example, wherein a heterodimerization domain isincorporated into the fusion protein so as the construct an asymmetricfusion protein. For example if an immunoglobulin Fc region isincorporated into the fusion protein the CH3 domain can be used ashomodimerization domain, or the CH3 dimer interface region can bemutated so as to enable heterodimerization.

In some embodiments, the fusion protein contains the DR5BDs at oppositeends. For example the DR5BDs are located on both the amino-terminal(N-terminal) portion of the fusion protein and the carboxy-terminal(C-terminal) portion of the fusion protein. In other embodiments, allthe DR5BDs reside on the same end of the fusion protein. For example,DR5BDs reside on either the amino or carboxyl terminal portions of thefusion protein.

In some embodiments, the fusion protein contains an immunoglobulin Fcregion. In some embodiments, the immunoglobulin Fc region is an IgGisotype selected from the group consisting of IgG1 isotype, IgG2isotype, IgG3 isotype, and IgG4 subclass.

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment thereof is an IgG isotype. For example, theimmunoglobulin Fc region of the fusion protein is of human IgG1 isotype,having an amino acid sequence:

(SEQ ID NO: 1)

APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMHEALHNHYTQK SLSLSPGK 

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment thereof comprises a human IgG1 polypeptide sequence thatis at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQID NO: 1.

In some embodiments, the human IgG1 Fc region is modified at amino acidAsn297 (Boxed, Kabat Numbering) to prevent to glycosylation of thefusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In someembodiments, the Fc region of the fusion protein is modified at aminoacid Leu235 (Boxed, Kabat Numbering) to alter Fc receptor interactions,e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, theFc region of the fusion protein is modified at amino acid Leu234 (Boxed,Kabat Numbering) to alter Fc receptor interactions, e.g., Leu234Ala(L234A). In some embodiments, the Fc region of the fusion protein isaltered at both amino acid 234 and 235, e.g., Leu234Ala and Leu235Ala(L234A/L235A) or Leu234Val and Leu235Ala (L234V/L235A). In someembodiments, the Fc region of the fusion protein is altered at Gly235 toreduce Fc receptor binding. For example, wherein Gly235 is deleted fromthe fusion protein. In some embodiments, the human IgG1 Fc region ismodified at amino acid Gly236 to enhance the interaction with CD32A,e.g., Gly236Ala (G236A). In some embodiments, the human IgG1 Fc regionis lacks Lys447 (EU index of Kabat et at 1991 Sequences of Proteins ofImmunological Interest).

In some embodiments, the Fc region of the fusion protein is altered atone or more of the following positions to reduce Fc receptor binding:Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298(S298), Asn297 (N297), Asn325 (N325) or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N),Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E) or Ala327Ser(A327S). In preferred embodiments, modifications within the Fc regionreduce binding to Fc-receptor-gamma receptors while have minimal impacton binding to the neonatal Fc receptor (FcRn).

In some embodiments, the Fc region of the fusion protein is lacking anamino acid at one or more of the following positions to reduce Fcreceptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). Inthese embodiments, Fc deletion of these three amino acids reduces thecomplement protein C1q binding.

(SEQ ID NO: 2) PAPGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQDWLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLPPSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK

In some embodiments, the fusion or immunologically active fragmentthereof comprises a human IgG2 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG2 isotype, havingan amino acid sequence:

(SEQ ID NO: 3) PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD

PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVEWESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK

In some embodiments, the fusion or immunologically active fragmentthereof comprises a human IgG2 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the human IgG2 Fc region is modified at amino acidAsn297 (Boxed, to prevent to glycosylation of the antibody, e.g.,Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the humanIgG2 Fc region is lacks Lys447 (EU index of Kabat et at 1991 Sequencesof Proteins of Immunological Interest).

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG3 isotype, havingan amino acid sequence:

(SEQ ID NO: 4) PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFKWYV

APIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAVEWESSGQPEN NYNTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNIFSCSVMH

In some embodiments, the antibody or immunologically active fragmentthereof comprises a human IgG3 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the human IgG3 Fc region is modified at amino acidAsn297 (Boxed, Kabat Numbering) to prevent to glycosylation of theantibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In someembodiments, the human IgG3 Fc region is modified at amino acid 435 toextend the half-life, e.g., Arg435His (R435H). In some embodiments, thehuman IgG3 Fc region is lacks Lys447 (EU index of Kabat et at 1991Sequences of Proteins of Immunological Interest).

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG4 isotype, havingan amino acid sequence:

(SEQ ID NO: 5)

SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMHEALHNHYTQK SLSLSLGK

In some embodiments, the antibody or immunologically active fragmentthereof comprises a human IgG4 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG4 isotype, havingan amino acid sequence:

(SEQ ID NO: 127) PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV

SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMHEALHNHYTQK SLSLSLGK

In some embodiments, the antibody or immunologically active fragmentthereof comprises a human IgG4 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 127.

In other embodiments, the human IgG4 Fc region is modified at amino acid235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In someembodiments, the human IgG4 Fc region is modified at amino acid Asn297(Boxed, Kabat Numbering) to prevent to glycosylation of the antibody,e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, thehuman IgG4 Fc region is lacks Lys447 (EU index of Kabat et at 1991Sequences of Proteins of Immunological Interest).

In some embodiments, the human IgG Fc region is modified to enhance FcRnbinding. Examples of Fc mutations that enhance binding to FcRn areMet252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively)(Kabat numbering, Dall'Acqua et at 2006, J. Biol Chem Vol. 281(33)23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et at2010 Nature Biotech, Vol. 28(2) 157-159), or Met252Ile, Thr256Asp,Met428Leu (M2521, T256D, M428L, respectively), (EU index of Kabat et at1991 Sequences of Proteins of Immunological Interest).

In some embodiments where the fusion protein of the disclosure includesan Fc polypeptide, the Fc polypeptide is mutated or modified. In theseembodiments the mutated or modified Fc polypeptide includes thefollowing mutations: Met252Tyr and Met428Leu or Met252Tyr and Met428Val(M252Y, M428L, or M252Y, M428V) using the Kabat numbering system.

In some embodiments, the human IgG Fc region is modified to alterantibody-dependent cellular cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC), e.g., the amino acidmodifications described in Natsume et al., 2008 Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al.,2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010,Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. EnzymeRegul., 48: 152-164; Alegre et al, 1992 J Immunol, 148: 3461-3468;Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11. Examples ofmutations that enhance ADCC include modification at Ser239 and Ile332,for example Ser239Asp and Ile332Glu (S239D, I332E). Examples ofmutations that enhance CDC include modifications at Lys326 and Glu333.In some embodiments the Fc region is modified at one or both of thesepositions, for example Lys326Ala and/or Glu333Ala (K326A and E333A)using the Kabat numbering system.

In some embodiments, the human IgG Fc region is modified to induceheterodimerization. For example, having an amino acid modificationwithin the CH3 domain at Thr366, which when replaced with a more bulkyamino acid, e.g., Try (T366W), is able to preferentially pair with asecond CH3 domain having amino acid modifications to less bulky aminoacids at positions Thr366, Leu368, and Tyr407, e.g., Ser, Ala and Val,respectively (T366S/L368A/Y407V). Heterodimerization via CH3modifications can be further stabilized by the introduction of adisulfide bond, for example by changing Ser354 to Cys (S354C) and Y349to Cys (Y349C) on opposite CH3 domains (Reviewed in Carter, 2001 Journalof Immunological Methods, 248: 7-15).

In some embodiments, the human IgG Fc region is modified to preventdimerization. In these embodiments, the fusion proteins of the presentdisclosure are monomeric. For example modification at residue Thr366 toa charged residue, e.g. Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu(T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3dimerization.

In some embodiments, the Fc region of the fusion protein is altered atone or more of the following positions to reduce Fc receptor binding:Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298(S298), Asn297 (N297), Asn325 (N325) or Ala327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N),Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E) or Ala327Ser(A327S). In preferred embodiments, modifications within the Fc regionreduce binding to Fc-receptor-gamma receptors while have minimal impacton binding to the neonatal Fc receptor (FcRn).

In some embodiments, the fusion protein contains a polypeptide derivedfrom an immunoglobulin hinge region. The hinge region can be selectedfrom any of the human IgG subclasses. For example the fusion protein maycontain a modified IgG1 hinge having the sequence of EPKSSDKTHTCPPC (SEQID NO: 6), where in the Cys220 that forms a disulfide with theC-terminal cysteine of the light chain is mutated to serine, e.g.,Cys220Ser (C220S). In other embodiments, the fusion protein contains atruncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 7).

In some embodiments, the fusion protein has a modified hinge from IgG4,which is modified to prevent or reduce strand exchange, e.g., Ser228Pro(S228P), having the sequence ESKYGPPCPPC (SEQ ID NO: 8). In someembodiments, the fusion protein contains linker polypeptides. In otherembodiments, the fusion protein contains linker and hinge polypeptides.

In some embodiments, the fusion proteins of the present disclosure lackor have reduced Fucose attached to the N-linked glycan-chain at N297.There are numerous ways to prevent fucosylation, including but notlimited to production in a FUT8 deficient cell line; addition inhibitorsto the mammalian cell culture media, for example Castanospermine; andmetabolic engineering of the production cell line.

In some embodiments, the DR5BD is engineered to eliminate recognition bypre-existing antibodies found in humans. In some embodiments, singledomain antibodies of the present disclosure are modified by mutation ofposition Leu11, for example Leu11Glu (L11E) or Leu11Lys (L11K). In otherembodiments, single domain antibodies of the present disclosure aremodified by changes in carboxy-terminal region, for example the terminalsequence consists of GQGTLVTVKPGG (SEQ ID NO: 9) or GQGTLVTVEPGG (SEQ IDNO: 10) or modification thereof. In some embodiments, the single domainantibodies of the present disclosure are modified by mutation ofposition 11 and by changes in carboxy-terminal region.

In some embodiments, the DR5BDs of the fusion proteins of the presentdisclosure are operably linked via amino acid linkers. In someembodiments, these linkers are composed predominately of the amino acidsGlycine and Serine, denoted as GS-linkers herein. The GS-linkers of thefusion proteins of the present disclosure can be of various lengths, forexample 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 aminoacids in length.

In some embodiments, the GS-linker comprises an amino acid sequenceselected from the group consisting of GGSGGS, i.e., (GGS)₂ (SEQ ID NO:11); GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 12); GGSGGSGGSGGS, i.e., (GGS)₄(SEQ ID NO: 13); and GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO: 14).

In some embodiments, the multivalent TNFRSF binding fusion protein istetravalent. In some embodiments, the tetravalent TNFRSF binding fusionprotein has the following structure: VHH-Linker-VHH-Linker-Hinge-Fc,where the VHH is a humanized or fully human VHH sequence that binds atleast DR5.

In some embodiments, the multivalent TNFRSF binding fusion protein istetravalent. In some embodiments, the tetravalent TNFRSF binding fusionprotein has the following structure: DR5BD-Linker-DR5BD-Linker-Hinge-Fc,where the DR5BD is a humanized or fully human VHH sequence.

In some embodiments, the multivalent TNFRSF binding fusion protein ishexavalent. In some embodiments, the hexavalent TNFRSF binding fusionprotein has the following structure:VHH-Linker-VHH-Linker-VHH-Linker-Hinge-Fc, where the VHH is a humanizedor fully human VHH sequence that binds at least DR5.

In some embodiments, the multivalent TNFRSF binding fusion protein ishexavalent. In some embodiments, the hexavalent TNFRSF binding fusionprotein has the following structure:DR5BD-Linker-DR5BD-Linker-DR5BD-Linker-Hinge-Fc, where the DR5BD is ahumanized or fully human VHH sequence.

In some embodiments, the multivalent fusion proteins targeting DR5 ofthe present disclosure are operably linked via amino acid linkers. Insome embodiments, these linkers are composed predominately of the aminoacids Glycine and Serine, denoted as GS-linkers herein. The GS-linkersof the fusion proteins of the present disclosure can be of variouslengths, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 amino acids in length.

In some embodiments, the GS-linker comprises an amino acid sequenceselected from the group consisting of GGSGGS, i.e., (GGS)₂ (SEQ ID NO:11); GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 12); GGSGGSGGSGGS, i.e., (GGS)₄(SEQ ID NO: 13); and GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO: 14).

In some embodiments, the multivalent DR5 binding fusion protein istetravalent. In some embodiments, the tetravalent DR5 binding fusionprotein has the following structure: VHH-Linker-VHH-Linker-Hinge-Fc,where the VHH is a humanized or fully human VHH sequence. In someembodiments, the VHH sequence is selected from the group consisting ofSEQ ID NO: 15-91. In some embodiments, the tetravalent DR5 bindingfusion protein comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 92-118.

In some embodiments, the multivalent DR5 binding fusion protein ishexavalent. In some embodiments, the hexavalent DR5 binding fusionprotein has the following structure:VHH-Linker-VHH-Linker-VHH-Linker-Hinge-Fc, where the VHH is a humanizedor fully human VHH sequence. In some embodiments, the VHH sequence isselected from the group consisting of SEQ ID NO: 15-91. In someembodiments, the hexavalent DR5 binding fusion protein comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:119-124.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is schematic of exemplary multivalent and multispecific fusionproteins of the present disclosure.

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G are a series of graphsdemonstrating the binding of representative DR5 VHHs or humanizedvariants thereof to either human DR5 (FIGS. 2A, 2B, 2E, 2F, and 2G) andcyno DR5 (FIGS. 2C and 2D). FIGS. 2A, 2B, 2E, 2F, and 2G demonstrate thebinding of some VHHs and humanized VHHs binding to human DR5 as assessedby flow cytometry on DR5 expressing CHO cells. FIGS. 2C and 2Ddemonstrate the binding of VHHs and humanized VHHs binding to cyno DR5as assessed by ELISA using recombinant cyno DR5. In FIGS. 2A, B, C, Dand E, the DR5 targeting fusion proteins used were bivalent, and formatsused were VHH-Fc or humanized (hz) hzVHH-Fc. In FIGS. 2F and 2G,humanized tetravalent (VHH-linker-VHH-Fc) DR5 targeting Fc-fusionproteins were used.

FIGS. 3A, 3B, and 3C are a series of graphs demonstrating the directapoptosis inducing capacity of DR5 targeting fusion proteins of thepresent disclosure. In all assays, the Colo205 cells were used and theDR5 targeting VHH was H10 formatted as (A) H10-Fc (bivalent), (B)H10-linker-H10-Fc (tetravalent), or (C) H10-linker-H10-linker-H10-Fc(hexavalent). FIG. 3A is a graph demonstrating the enhanced apoptosisinducing capacity of a bivalent DR5 targeting fusion protein when acrosslinking agent is used. FIG. 3B is a graph demonstrating theenhanced apoptosis inducing capacity of a tetravalent DR5 targetingfusion protein compared to a bivalent DR5 targeting fusion protein. FIG.3C is a graph demonstrating the enhanced apoptosis inducing capacity ofa tetravalent and furthermore hexavalent DR5 targeting fusion proteincompared to a bivalent DR5 targeting fusion protein and TRAIL.

FIG. 4A is a graph demonstrating the ability of a hexavalent DR5targeting fusion protein to induce apoptosis of the resistant cell linePanc-1. Colo205 is shown for comparison. H10 DR5 targeting VHH is shown.

FIG. 4B is a graph demonstrating the enhanced sensitivity of Panc-1 to atetravalent DR5 targeting fusion protein when doxorubicin is added. TheDR5 targeting VHH shown is humanized F03 (hzF03), formatted ashzF03-linker-hzF03-Fc.

FIG. 5 is a graph demonstrating the anti-tumor activity of tetravalentDR5 targeting fusion proteins of the present disclosure in a murinetumor xenograft model with Colo-205 cells. Fusion proteins were dosed at1 mg/kg weekly for 4 weeks via IV administration. Dosing began whentumors reached approximately 300 mm³.

FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, and 6J are a series of graphsdemonstrating direct cell death inducing capacity of some of thetetravalent DR5 targeting fusion proteins of the present disclosurecompared to TAS266 (a tetravalent DR5 nanobody described in PCTPublication No. WO 2011/098520A1) on various cancer cell lines (FIGS. 6Aand 6B) Colo-205, Panc-1 (FIGS. 6C and 6J), JL-1 (FIG. 6D), HCT-116(FIG. 6E), NCI-H28 (FIG. 6F), NCI-H460 (FIG. 6G), HT-29 (FIG. 6H), andMSTO-211H (FIG. 6I). In FIGS. 6A-6D, the DR5 targeting VHH is ahumanized variant of 1F5 (hz1F5) formatted as hzVHH-linker-hzVHH-Fc. InFIGS. 6E-6J, the DR5 targeting VHH is a humanized variant of either 1F2(hz1F2) or 2C6 (hz2C6) formatted as hzVHH-linker-hzVHH-Fc variants.

FIG. 7A is a graph demonstrating the differences in autoantibodyrecognition of TAS266, (a tetravalent DR5 nanobody described in PCTPublication No. WO 2011/098520A1) and humanized tetravalent 1F5(Tet-hz1F5v5) of present disclosure. This graph depicts the results fromthe serum of 45 human donors. Autoantibodies containing either a kappaor lambda light chain were detected in separate assays using therespective anti-human Ig Kappa or anti-human Ig Lambda HRP-conjugatedsecondary antibodies. Data are normalized to positive control of an IgGantibody having either a lambda or kappa light chain, respectively.TAS266 displays significant autoantibody recognition while autoantibodyrecognition of Tet-hz1F5v5 is reduced to that of IgG control background.

FIG. 7B is a graph that demonstrates that pooled serum from multiplehuman donors (IVIG, Gamunex®-C, Grifols) contains some IgG antibodiesthat recognize single domain antibodies (sdAb) including TAS266.

FIG. 7C is a graph that demonstrates that recognition of TAS266 byautoantibodies within IVIG induces apoptosis of primary humanhepatocytes.

FIGS. 8A and 8B are a series of graphs demonstrating the autoantibodyrecognition-dependent hepatotoxicity of TAS266, but not Tet-hz1F5v5, onHepRG™ the terminally differentiated hepatic cells derived from ahepatic progenitor cell line. In FIG. 8A, apoptosis was monitored usinga caspase-3/7-specific fluorogenic substrate with IncuCyte Zoom livecell imager (Essen Biosciences), data shown is at 48 hours. In FIG. 8B,apoptosis was monitored after 48 hours using a CellTiter Glo assay(Promega). IVIG (Gamunex®-C, Grifols) was used an sdAb-directedautoantibody containing antibody pool.

FIGS. 9A, 9B, 9C, and 9D are a series of graphs demonstrating theautoantibody recognition-dependent hepatoxicity of TAS266, but not thetetravalent DR5 targeting fusion proteins of the present disclosure.HepRG™ cells were used as a surrogate for human hepatocytes. IVIG(Gamunex®-C, Grifols) was used an sdAb-directed autoantibody containingantibody pool. FIGS. 9A and 9C depict independent 48 hour assays anddemonstrate that TAS266 induces hepatotoxicity when crosslinked byautoantibodies. Moderate hepatotoxicity was observed when a crosslinkinganti-human Fc secondary was added to the tetravalent DR5 targetingfusion proteins of the present disclosure, hz1F5, hz1F2 or hz2C6,formatted as hzVHH-linker-hzVHH-Fc. Cell viability was assessed byCellTiter Glo (Promega). FIG. 9D depicts the reduction autoantibodyrecognition-dependent hepatotoxicity of TAS266 when it is modified atamino acid positions Leu11 and the C-terminal region of each of the fourDR5 sdAbs (FIX-TAS266, SEQ ID NO: 126). This data demonstrate thathepatocytoxicity of FIX-266 in the presence of IVIG is reduced to thatof TAS266 in the absence of IVIG. HepRG cell viability was assessed byCellTiter Glo (Promega). FIG. 9B depicts the kinetics of autoantibodyrecognition-dependent hepatotoxicity of TAS266 as well as the secondaryantibody crosslinking-dependent hepatotoxicity of Tet-hz1F5v6. Apoptosiswas monitored over a 46 hour period using a caspase-3/7-specificfluorogenic substrate with IncuCyte Zoom live cell imager (EssenBiosciences). Tetravalent DR5 targeting fusion proteins of the presentdisclosure do not induce hepatotoxicity in the presence or absence ofsdAb-directed autoantibody containing antibodies.

DETAILED DESCRIPTION

The disclosure provides molecules that specifically engage deathreceptor 5 (DR5), a member of the TNF receptor superfamily (TNFRSF).More specifically this disclosure relates to multivalent molecules thatbind at least DR5. These multivalent TNFRSF binding fusion proteinscomprise two or more TNFRSF binding domains (DR5BDs), where at least oneDR5BD binds DR5. These molecules are referred to herein as DR5-targetingmolecules.

These DR5-targeting molecules include at least one copy of asingle-domain antibody (sdAb) sequence that specifically binds DR5. Insome embodiments, the DR5-targeting molecules include two or more copiesof a sdAb that specifically binds DR5, for example, three or more, fouror more, five or more, or six or more copies of a sdAb that specificallybinds DR5.

A single-domain antibody (sdAb) is an antibody fragment consisting of asingle monomeric variable antibody domain that is able to bindselectively to a specific antigen. With a molecular weight of only 12-15kDa, single-domain antibodies are much smaller than common antibodies(150-160 kDa) which are composed of two heavy protein chains and twolight chains, and even smaller than Fab fragments (˜50 kDa, one lightchain and half a heavy chain) and single-chain variable fragments (˜25kDa, two variable domains, one from a light and one from a heavy chain).

Single domain antibodies are antibodies whose complementary determiningregions are part of a single domain polypeptide. Examples include, butare not limited to, heavy chain antibodies, antibodies naturally devoidof light chains, single domain antibodies derived from conventional4-chain antibodies, engineered antibodies and single domain scaffoldsother than those derived from antibodies. Single domain antibodies maybe derived from any species including, but not limited to mouse, human,camel, llama, goat, rabbit, and/or bovine. In some embodiments, a singledomain antibody as used herein is a naturally occurring single domainantibody known as heavy chain antibody devoid of light chains. Forclarity reasons, this variable domain derived from a heavy chainantibody naturally devoid of light chain is known herein as a VHH todistinguish it from the conventional VH of four chain immunoglobulins.Such a VHH molecule can be derived from antibodies raised in Camelidaespecies, for example in camel, llama, dromedary, alpaca and guanaco.Other species besides Camelidae may produce heavy chain antibodiesnaturally devoid of light chain; such VHHs are within the scope of thedisclosure.

A single-domain antibody can be obtained by immunization of dromedaries,camels, llamas, alpacas or sharks with the desired antigen andsubsequent isolation of the mRNA coding for heavy-chain antibodies. Byreverse transcription and polymerase chain reaction, a gene library ofsingle-domain antibodies containing several million clones is produced.Screening techniques like phage display and ribosome display help toidentify the clones binding the antigen. (See e.g., Arbabi Ghahroudi,M.; Desmyter, A.; et al. (1997). “Selection and identification of singledomain antibody fragments from camel heavy-chain antibodies”. FEBSLetters 414 (3): 521-526.)

A different method uses gene libraries from animals that have not beenimmunized beforehand. Such naïve libraries usually contain onlyantibodies with low affinity to the desired antigen, making it necessaryto apply affinity maturation by random mutagenesis as an additionalstep. (Saerens, D.; et al. (2008). “Single-domain antibodies as buildingblocks for novel therapeutics”. Current Opinion in Pharmacology 8 (5):600-608.)

When the most potent clones have been identified, their DNA sequence isoptimized, for example to improve their stability towards enzymes.Another goal is humanization to prevent immunological reactions of thehuman organism against the antibody. Humanization is unproblematicbecause of the homology between camelid VHH and human VH fragments. (Seee.g., Saerens, et al., (2008). “Single-domain antibodies as buildingblocks for novel therapeutics”. Current Opinion in Pharmacology 8 (5):600-608.) The final step is the translation of the optimizedsingle-domain antibody in E. coli, Saccharomyces cerevisiae or othersuitable organisms.

Single domain antibody fragments are also derived from conventionalantibodies. In some embodiments, single-domain antibodies can be madefrom common murine or human IgG with four chains. (Holt, L. J.; et al.(2003). “Domain antibodies: proteins for therapy”. Trends inBiotechnology 21 (11): 484-490.) The process is similar, comprising genelibraries from immunized or naïve donors and display techniques foridentification of the most specific antigens. A problem with thisapproach is that the binding region of common IgG consists of twodomains (VH and VL), which tend to dimerize or aggregate because oftheir lipophilicity. Monomerization is usually accomplished by replacinglipophilic by hydrophilic amino acids, but often results in a loss ofaffinity to the antigen. (See e.g., Borrebaeck, C. A. K.; Ohlin, M.(2002). “Antibody evolution beyond Nature”. Nature Biotechnology 20(12): 1189-90.) If affinity can be retained, the single-domainantibodies can likewise be produced in E. coli, S. cerevisiae or otherorganisms.

Monovalent single domain antibodies can be made multivalent via severalmethods. For example the cDNA encoding a first sdAb can be geneticallyfused to a linker encoding DNA sequence followed by a second cDNAencoding an sdAb and so forth and so on. Alternatively, the cDNAencoding an sdAb can be fused to cDNA encoding a second protein orfragment thereof that naturally multimerizes or is engineered tomultimerize. For example, fusion of an sdAb to an IgG Fc region willdimerize the sdAb. Wherein a tandem sdAb encoding constructed is linkedto an Fc encoding construct the resultant fusion protein once expressedwill be tetravalent. Wherein a construct that encodes three sdAbs islinked to an Fc encoding construct the resultant fusion protein onceexpressed will be hexavalent. This disclosure contemplates the use ofthe additional multimerization domains, including collagenhomotrimerization and heterotrimerization domains, leucine zipperdomains, p53 tetramerization domains, c-Jun:Fos heterodimeric peptidesequences, cartilage oligomeric matrix protein (COMP48), trimericadiponectin, trimeric surfactant protein D, and/or synapticacetylcholinesterase tetramer.

Death Receptor 5 (TRIAL-R2, TNFRSF10B) Targeting

The TNF-related apoptosis-inducing ligand (TRAIL) evolved to playcritical roles in mammalian development and host defense by selectivelyeradicating unwanted, infected and malignant cells from healthy cellpopulations. On binding the TNF receptor family members DR4 or DR5,TRAIL induces cell death via caspase-dependent apoptosis. DR5(TNFRSF10B) appears to be the primary receptor on tumor cells thatfacilitates the observed tumor biased activity of the TRAIL pathway. DR5is activated by the natural ligand TRAIL, which brings three DR5receptors within close proximity thereby activating intracellularcaspase-8 and initiating activation of other death-inducing caspases,such as caspases-9 and caspases-3. Thus initiation of this cell deathpathway requires clustering of DR5 receptors for efficient cell death.

Efforts to clinically exploit the TRAIL pathway for cancer therapyrelied upon a recombinant version of the natural ligand TRAIL andantibodies specific for DR5. Antibody agonists targeting DR5 required acrosslinking agent in preclinical in vitro experiments. This was due tothe fact the conventional antibodies resulted in clustering of only twoDR5 receptors (one per each heavy and light chain). Two DR5 receptorsare insufficient to activate the cell death pathway thus the need for acrosslinking agent. Surprisingly in vivo administration of DR5 targetingantibodies in pre-clinical mouse models of human cancers showedsignificant activity in a wide variety of tumor types. This activity waslater shown to be dependent on mouse FcgammaR (FcγR) receptors. Clinicalstudies in humans failed to reproduce the robust responses seen in thesepre-clinical mouse models. The lack of activity in humans ishypothesized to be due to insufficient antibody crosslinking. This maybe due to differences in serum IgG, FcgammaR (FcγR) and or TRAILconcentrations between immune compromised mice and human cancerpatients.

The present disclosure provides multivalent fusion proteins targetingDR5 that are capable of potently agonizing DR5 signaling mediatingdirect cell death. The fusion proteins of the present disclosure can betrivalent, tetravalent, pentavalent, or hexavalent. Importantly, thefusion proteins of the present disclosure are capable of elicitingapoptosis of DR5 expressing cells independently of exogenouscrosslinking agents.

In some embodiments, the fusion proteins of the present disclosureincorporate a DR5BD that binds DR5. In preferred embodiments, the DR5binding DR5BD does not bind DR4, decoy R1, decoy R2, Osteopontin, or anyother TNFRSF member. In preferred embodiments the DR5 binding DR5BDbinds human and cynomolgus monkey DR5. In some embodiments, the DR5binding DR5BD blocks the interaction of DR5 and its ligand TRAIL. Inother embodiments, the DR5 binding DR5BD does not block the interactionof DR5 and its ligand TRAIL. In some embodiments, the fusion protein ofthe present disclosure incorporates multiple DR5 binding DR5BDs thatrecognize distinct epitopes on DR5. In some embodiments, the fusionprotein of the present disclosure incorporates multiple DR5 bindingDR5BDs, wherein some DR5BDs block the DR5-TRAIL interaction and other donot block the DR5-TRAIL interaction. In preferred embodiments, DR5targeting fusion proteins of the present disclosure induce direct celldeath of tumor cells. The DR5 targeting fusion proteins of the presentdisclosure have utility in treating tumors of both hematologic and solidin nature.

Exemplary DR5 Binding sdAbs

DR5 VHH (llama-derived) and humanized sequences are shown below, and theCDR sequences are shown below each sequence. In some embodiments, theDR5 binding sdAb is fused to an IgG Fc region and in these embodimentsthe fusion protein is bivalent having two DR5 binding domains permolecule. In some embodiments, two DR5 binding sdAbs (2×) are fused toan IgG Fc region and in these embodiments the fusion protein istetravalent having four DR5 binding domains per molecule. In someembodiments, three DR5 binding sdAbs (3×) are fused to an IgG Fc regionand in these embodiments the fusion protein is hexavalent having six DR5binding domains per molecule.

1F5 (SEQ ID NO: 15) QVQLVQSGGGLVQAGDSLRLSCAASGLTFPNYGMGWFRQAPGEEREFLAVIYWSGGTVFYADSVKGRFTISRDAAKNMVYLQMNSLKSDDTAVYYCAVTIRGAATQTWKYDYWGRGTQVTVSS (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 129) CDR2: VIYWSGGTVF (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW hz1F5v1 (SEQ ID NO: 16)EVQLLESGGGLVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVSAIYWSGGTVYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVSS (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 131) CDR2: AIYWSGGTVY (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW hz1F5v1opt (SEQ ID NO: 17)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVSAIYWSGGTVYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKPGG (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 131) CDR2: AIYWSGGTVY (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW hz1F5v1opt1 (SEQ ID NO: 18)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVSAIYWSGGTVYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKP (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 131) CDR2: AIYWSGGTVY (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW hz1F5v2 (SEQ ID NO: 19)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKEREFVSAIYWSGGTVYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTQVTVKP (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 131) CDR2: AIYWSGGTVY (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW hz1F5v1DS (SEQ ID NO: 20)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVCAIYWSGGTVYYAESVKGRFTCSRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKPGG (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 131) CDR2: AIYWSGGTVY (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW hz1F5v3 (SEQ ID NO: 85)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFVSAIYWSGGTVFYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKP (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 132) CDR2: AIYWSGGTVF (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW hz1F5v4 (SEQ ID NO: 86)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFLAVIYWSGGTVFYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKP (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 129) CDR2: VIYWSGGTVF (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW hz1F5v5 (SEQ ID NO: 87)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFVSAIYWSGGTVYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKP (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 131) CDR2: AIYWSGGTVY (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW hz1F5v6 (SEQ ID NO: 88)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFLAVIYWSGGTVYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKP (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 133) CDR2: VIYWSGGTVY (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW hz1F5v7 (SEQ ID NO: 89)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFVSAIYWSGGTVYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKP (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 131) CDR2: AIYWSGGTVY (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW hz1F5v8 (SEQ ID NO: 90)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFLAVIYWSGGTVYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKP (SEQ ID NO: 128) CDR1: SGLTFPNYGM(SEQ ID NO: 133) CDR2: VIYWSGGTVY (SEQ ID NO: 130)CDR3: AVTIRGAATQTWKYDYW 2C6 (SEQ ID NO: 21)QVQLVQSGGGLVQAGGSLRLTCTASGRTVSNYAMGWFRQTPGKDREFVAALNWSGDTTSYADSVRGRFTISRDNTRNTVYLQMDSLKREDTAVYYCAAAQSFRRGGAPYGDNYWGQGTQVTVSS (SEQ ID NO: 134) CDR1: SGRTVSNYAM(SEQ ID NO: 135) CDR2: ALNWGGDTTS (SEQ ID NO: 136)CDR3: AAAQSFRRGGAPYGDNYW hz2C6v1 (SEQ ID NO: 22)EVQLLESGGGLVQPGGSLRLSCAASGRTVSNYAMSWFRQAPGKGLEFVSALNWGGDTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAAQSFRRGGAPYGDNYWWGQGTLVTVSS (SEQ ID NO: 134) CDR1: SGRTVSNYAM(SEQ ID NO: 137) CDR2: ALNWGGDTTY (SEQ ID NO: 136)CDR3: AAAQSFRRGGAPYGDNYW hz2C6v1opt (SEQ ID NO: 23)EVQLLESGGGEVQPGGSLRLSCAASGRTVSNYAMSWFRQAPGKGLEFVSALNWGGDTTYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAAAQSFRRGGAPYGDNYWGQGTLVTVKPGG (SEQ ID NO: 134) CDR1: SGRTVSNYAM(SEQ ID NO: 137) CDR2: ALNWGGDTTY (SEQ ID NO: 136)CDR3: AAAQSFRRGGAPYGDNYW hzC06v2 (SEQ ID NO: 91)EVQLLESGGGEVQPGGSLRLSCAASGRTVSNYAMGWFRQAPGKDREFVSALNWGGDTTYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAAAQSFRRGGAPYGDNYWGQGTLVTVKP (SEQ ID NO: 134) CDR1: SGRTVSNYAM(SEQ ID NO: 137) CDR2: ALNWGGDTTY (SEQ ID NO: 136)CDR3: AAAQSFRRGGAPYGDNYW C12 (SEQ ID NO: 24)EVQLVQSGGGLVQAGDSLRLSCAASGRALTGYHMAWFRQAPGKEREFVTYGIWDRAGAAYADSVKGRFTMSRDNAKNTVYLQMNNLKTEDTAVYYCAASMAVRTYYSPRSYDSWGQGTQVTVSS (SEQ ID NO: 138) CDR1: SGRALTGYHMAW(SEQ ID NO: 139) CDR2: YGIWDRAGAA (SEQ ID NO: 140)CDR3: ASMAVRTYYSPRSYDSW hzC12v2 (SEQ ID NO: 25)EVQLLESGGGLVQPGGSLRLSCAASGRALTGYHMSWFRQAPGKGREFVSYGIWDRAGAAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASMAVRTYYSPRSYDSWGQGTLVTVSS (SEQ ID NO: 141) CDR1: SGRALTGYHMSW(SEQ ID NO: 139) CDR2: YGIWDRAGAA (SEQ ID NO: 140)CDR3: ASMAVRTYYSPRSYDSW hzC12v3 (SEQ ID NO: 26)EVQLLESGGGLVQPGGSLRLSCAASGRALTGYHMSWFRQAPGKGLEFVSYGIWDRAGAAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASMAVRTYYSPRSYDSWGQGTLVTVSS (SEQ ID NO: 141) CDR1: SGRALTGYHMSW(SEQ ID NO: 139) CDR2: YGIWDRAGAA (SEQ ID NO: 140)CDR3: ASMAVRTYYSPRSYDSW 1F2 (SEQ ID NO: 27)EVQLVQSGGGLVQAGGSLRLSCAASGSTFSSLDMGWFRQAPGKERAFVAAISRSGDNIYYAESVKGRFTISRDNAENTTYLQMNSLKPEDSAVYYCAVDSQPTYSGGVYYPRYGMDVWGQGTQVTVSS (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 144)CDR3: AVDSQPTYSGGVYYPRYGMDVW hz1F2v2 (SEQ ID NO: 29)EVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGMDVWGQGTLVTVSS (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 144)CDR3: AVDSQPTYSGGVYYPRYGMDVW hz1F2v1 (SEQ ID NO: 30)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGMDVWGQGTLVTVKP (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 144)CDR3: AVDSQPTYSGGVYYPRYGMDVW hz1F2v2 (SEQ ID NO: 32)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDTQPTYSGGVYYPRYGMDVWGQGTLVTVKP (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 145)CDR3: AVDTQPTYSGGVYYPRYGMDVW hz1F2v3 (SEQ ID NO: 33)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDAQPTYSGGVYYPRYGMDVWGQGTLVTVKP (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 146)CDR3: AVDAQPTYSGGVYYPRYGMDVW hz1F2v4 (SEQ ID NO: 34)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVESQPTYSGGVYYPRYGMDVWGQGTLVTVKP (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 147)CDR3: AVESQPTYSGGVYYPRYGMDVW hz1F2v5 (SEQ ID NO: 35)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGYDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 148)CDR3: AVDSQPTYSGGVYYPRYGYDVW hz1F2v6 (SEQ ID NO: 36)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGDDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 148)CDR3: AVDSQPTYSGGVYYPRYGDDVW hz1F2v7 (SEQ ID NO: 37)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGLDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 149)CDR3: AVDSQPTYSGGVYYPRYGLDVW hz1F2-DS (SEQ ID NO: 38)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVCAISRSGDNIYYAESVKGRFTCSRDNAKNTLYLQMSSLRAEDTAVYYCAVESQPTYSGGVYYPRYGMDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 147)CDR3: AVESQPTYSGGVYYPRYGMDVW hz1F2-MA (SEQ ID NO: 39)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDAQPTYSGGVYYPRYGADVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 150)CDR3: AVDAQPTYSGGVYYPRYGADVW hz1F2-ME (SEQ ID NO: 40)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDAQPTYSGGVYYPRYGEDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 150)CDR3: AVDAQPTYSGGVYYPRYGEDVW hz1F2-MH (SEQ ID NO: 41)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDAQPTYSGGVYYPRYGHDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 151)CDR3: AVDAQPTYSGGVYYPRYGHDVW hz1F2-MN (SEQ ID NO: 42)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDAQPTYSGGVYYPRYGNDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 152)CDR3: AVDAQPTYSGGVYYPRYGNDVW hz1F2-MP (SEQ ID NO: 43)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDAQPTYSGGVYYPRYGPDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 153)CDR3: AVDAQPTYSGGVYYPRYGPDVW hz1F2-MQ (SEQ ID NO: 44)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDAQPTYSGGVYYPRYGQDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 154)CDR3: AVDAQPTYSGGVYYPRYGQDVW hz1F2-MR (SEQ ID NO: 45)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDAQPTYSGGVYYPRYGRDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 155)CDR3: AVDAQPTYSGGVYYPRYGRDVW hz1F2-MS (SEQ ID NO: 46)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDAQPTYSGGVYYPRYGSDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 156)CDR3: AVDAQPTYSGGVYYPRYGSDVW hz1F2-MT (SEQ ID NO: 47)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDAQPTYSGGVYYPRYGTDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 157)CDR3: AVDAQPTYSGGVYYPRYGTDVW hz1F2-MV (SEQ ID NO: 48)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVDAQPTYSGGVYYPRYGVDVWGQGTLVTVKPGG (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 158)CDR3: AVDAQPTYSGGVYYPRYGVDVW B04 (SEQ ID NO: 49)EVQLVQSGGGLVQAGGSLRLSCAASGRAFSNYALGWFRQAPGKEREFIAAINWNGENRYGVDSVKGRFTISRDNAQNMGYLQMNNLKPEDTAVYRCAAALSFRLGGEPYGDAYWGQGTQVTVSS (SEQ ID NO: 159) CDR1: SGRAFSNYALGW(SEQ ID NO: 160) CDR2: AINWNGENRY (SEQ ID NO: 161)CDR3: AAALSFRLGGEPYGDAYW hzB04v1 (SEQ ID NO: 50)EVQLLESGGGLVQPGGSLRLSCAASGRAFSNYAMSWFRQAPGKGLEFVSAINWNGENRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALSFRLGGEPYGDAYWGQGTLVTVSS (SEQ ID NO: 162) CDR1: SGRAFSNYAMSW(SEQ ID NO: 160) CDR2: AINWNGENRY (SEQ ID NO: 161)CDR3: AAALSFRLGGEPYGDAYW 5A04 (SEQ ID NO: 51)QVQLQESGGGLVQAGGSLRLSCVASGSIFTNNAMGWYRQAPGKQRDLVAQITMGGGITNYAPSMEGRFAISRDNAKSTVYLQMNNLKPEDTAVYYCNAEVKSADWGAYANYWGQGTQVTVSS (SEQ ID NO: 163) CDR1: SGSIFTNNAM(SEQ ID NO: 164) CDR2: QITMGGGITN (SEQ ID NO: 165)CDR3: NAEVKSADWGAYANYW hz5A04v1 (SEQ ID NO: 52)EVQLLESGGGLVQPGGSLRLSCAASGSIFTNNAMSWYRQAPGKGLELVSAITMGGGITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNAEVKSADWGAYANYWGQGTLVTVSS (SEQ ID NO: 163) CDR1: SGSIFTNNAM(SEQ ID NO: 166) CDR2: AITMGGGITY (SEQ ID NO: 165)CDR3: NAEVKSADWGAYANYW hz5A04v2 (SEQ ID NO: 53)EVQLLESGGGLVQPGGSLRLSCAASGSIFTNNAMSWYRQAPGKGRELVSQITMGGGITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNAEVKSADWGAYANYWGQGTLVTVSS (SEQ ID NO: 163) CDR1: SGSIFTNNAM(SEQ ID NO: 167) CDR2: QITMGGGITY (SEQ ID NO: 165)CDR3: NAEVKSADWGAYANYW F03 (SEQ ID NO: 54)QVQLQESGGGLVQAGGSLRLSCAASGRSISNYAMGWFRQAPGKEREFLAASVWNNGGNYYADSVKGRFTASRDDAKSTAYLQMSRLRPEDTGIYYCVVARTPETPITSARGANYWGQGTQVTVSS (SEQ ID NO: 168) CDR1: SGRSISNYAM(SEQ ID NO: 169) CDR2: ASVWNNGGNY (SEQ ID NO: 170)CDR3: VVARTPETPITSARGANYW hzF03v2 (SEQ ID NO: 55)EVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMGWFRQAPGKEREFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVSS (SEQ ID NO: 168) CDR1: SGRSISNYAM(SEQ ID NO: 169) CDR2: ASVWNNGGNY (SEQ ID NO: 170)CDR3: VVARTPETPITSARGANYW hzF03v1opt (SEQ ID NO: 56)EVQLLESGGGEVQPGGSLRLSCAASGRSISNYAMGWFRQAPGKEREFVSASVWNNGGNYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVKPGG (SEQ ID NO: 168) CDR1: SGRSISNYAM(SEQ ID NO: 169) CDR2: ASVWNNGGNY (SEQ ID NO: 170)CDR3: VVARTPETPITSARGANYW hzF03v2opt (SEQ ID NO: 57)EVQLLESGGGEVQPGGSLRLSCAASGRSISNYAMGWFRQAPGKEREFVSASVWNNGGNYYAESVKGRFTISRDDAKSTLYLQMSSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVKPGG (SEQ ID NO: 168) CDR1: SGRSISNYAM(SEQ ID NO: 169) CDR2: ASVWNNGGNY (SEQ ID NO: 170)CDR3: VVARTPETPITSARGANYW hzF03v3opt (SEQ ID NO: 58)EVQLLESGGGEVQPGGSLRLSCAASGRSISNYAMGWFRQAPGKEREFVSASVWNQGGNYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVKPGG (SEQ ID NO: 168) CDR1: SGRSISNYAM(SEQ ID NO: 171) CDR2: ASVWNQGGNY (SEQ ID NO: 170)CDR3: VVARTPETPITSARGANYW hzF03v4opt (SEQ ID NO: 59)EVQLLESGGGEVQPGGSLRLSCAASGRSISNYAMGWFRQAPGKEREFVSASVWNNAGNYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVKPGG (SEQ ID NO: 168) CDR1: SGRSISNYAM(SEQ ID NO: 172) CDR2: ASVWNNAGNY (SEQ ID NO: 170)CDR3: VVARTPETPITSARGANYW hzF03v5opt (SEQ ID NO: 60)EVQLLESGGGEVQPGGSLRLSCAASGRSISNYAMGWFRQAPGKEREFVSASVWNQGGNYYAESVKGRFTISRDDAKSTLYLQMSSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVKPGG (SEQ ID NO: 168) CDR1: SGRSISNYAM(SEQ ID NO: 171) CDR2: ASVWNQGGNY (SEQ ID NO: 170)CDR3: VVARTPETPITSARGANYW hzF03v6opt (SEQ ID NO: 61)EVQLLESGGGEVQPGGSLRLSCAASGRSISNYAMGWFRQAPGKEREFVSASVWNNAGNYYAESVKGRFTISRDDAKSTLYLQMSSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVKPGG (SEQ ID NO: 168) CDR1: SGRSISNYAM(SEQ ID NO: 172) CDR2: ASVWNNAGNY (SEQ ID NO: 170)CDR3: VVARTPETPITSARGANYW 3B7 (SEQ ID NO: 62)QVQLQESGGGSVQAGGSLTLSCAASGRAASDYAVGWFRQAPGKEREFVAACNWSGEDTVYAYIVKGRFTISRDNAGNTVSLRMSSLEPEDTAVYYCAAAPSFSRSVLDGNLSQIDYWGQGTQVTVSS (SEQ ID NO: 173) CDR1: SGRAASDYAV(SEQ ID NO: 174) CDR2: ACNWSGEDTV (SEQ ID NO: 175)CDR3: AAAPSFSRSVLDGNLSQIDYW hz3B7v2 (SEQ ID NO: 63)EVQLLESGGGLVQPGGSLRLSCAASGRAASDYAMSWFRQAPGKGLEFVSAINWGGEDTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAAPSFSRSVLDGNLSQIDYWGQGTLVTVSS (SEQ ID NO: 176) CDR1: SGRAASDYAM(SEQ ID NO: 177) CDR2: INWGGEDTV (SEQ ID NO: 175)CDR3: AAAPSFSRSVLDGNLSQIDYW 6G01 (SEQ ID NO: 64)QVQLVQSGGGLAQAGGSLRLSCVASGRTFTNYAMGWFRQAPGKEREFVAAINWSGDSTYHADSVKGRFTISRDNAKDSVYLQMTKLKPEDTADYYCASAESFSRGGLPYGMNYWGQGTQVTVSS (SEQ ID NO: 178) CDR1: SGRTFTNYAM(SEQ ID NO: 179) CDR2: AINWSGDSTY (SEQ ID NO: 180)CDR3: ASAESFSRGGLPYGMNYW hz6G01v1 (SEQ ID NO: 65)EVQLLESGGGLVQPGGSLRLSCAASGRTFTNYAMSWFRQAPGKGLEFVSAINWSGDSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASAESFSRGGLPYGMNYWGQGTLVTVSS (SEQ ID NO: 178) CDR1: SGRTFTNYAM(SEQ ID NO: 179) CDR2: AINWSGDSTY (SEQ ID NO: 180)CDR3: ASAESFSRGGLPYGMNYW hz6G01v1opt (SEQ ID NO: 66)EVQLLESGGGEVQPGGSLRLSCAASGRTFTNYAMSWFRQAPGKGLEFVSAINWSGDSTYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCASAESFSRGGLPYGMNYWGQGTLVTVKPGG (SEQ ID NO: 178) CDR1: SGRTFTNYAM(SEQ ID NO: 179) CDR2: AINWSGDSTY (SEQ ID NO: 180)CDR3: ASAESFSRGGLPYGMNYW H10 (SEQ ID NO: 67)QVQLVQSGGGLVQAGGSLTLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYADSVRGRFKNSKDNAKRTAYLQMNRLKPEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTQVTVSS (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 182) CDR2: AIRWDGVGAY (SEQ ID NO: 183)CDR3: ALPRRGDSELPSTVKEYGYW hzH10v3 (SEQ ID NO: 68)EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWEGVGAYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVKP (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 184) CDR2: AIRWEGVGAY (SEQ ID NO: 183)CDR3: ALPRRGDSELPSTVKEYGYW hzH10v2 (SEQ ID NO: 69)EVQLLESGGGLVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVSS (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 182) CDR2: AIRWDGVGAY (SEQ ID NO: 183)CDR3: ALPRRGDSELPSTVKEYGYW hzH10v1opt (SEQ ID NO: 70)EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVKPGG (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 182) CDR2: AIRWDGVGAY (SEQ ID NO: 183)CDR3: ALPRRGDSELPSTVKEYGYW hzH10-DS (SEQ ID NO: 71)EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVCAIRWEGVGAYYAESVKGRFTCSRDNAKNTLYLQMSSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVKPGG (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 184) CDR2: AIRWEGVGAY (SEQ ID NO: 183)CDR3: ALPRRGDSELPSTVKEYGYW hzH10v4opt (SEQ ID NO: 72)EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDAVGAYYAESVKGRFTISKDNAKRTLYLQMSSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVKPGG (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 185) CDR2: AIRWDAVGAY (SEQ ID NO: 183)CDR3: ALPRRGDSELPSTVKEYGYW hzH10v5opt (SEQ ID NO: 73)EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYAESVKGRFTISKDNAKRTLYLQMSSLRAEDTAVYYCALPRRGESELPSTVKEYGYWGQGTLVTVKPGG (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 182) CDR2: AIRWDGVGAY (SEQ ID NO: 186)CDR3: ALPRRGESELPSTVKEYGYW hzH10v6opt (SEQ ID NO: 74)EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYAESVKGRFTISKDNAKRTLYLQMSSLRAEDTAVYYCALPRRGDAELPSTVKEYGYWGQGTLVTVKPGG (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 182) CDR2: AIRWDGVGAY (SEQ ID NO: 187)CDR3: ALPRRGDAELPSTVKEYGYW hzH10v7opt (SEQ ID NO: 75)EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVKPGG (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 182) CDR2: AIRWDGVGAY (SEQ ID NO: 183)CDR3: ALPRRGDSELPSTVKEYGYW hzH10v8opt (SEQ ID NO: 76)EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWEGVGAYYAESVKGRFTISKDNAKRTLYLQMSSLRAEDTAVYYCALPRRGESELPSTVKEYGYWGQGTLVTVKPGG (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 184) CDR2: AIRWEGVGAY (SEQ ID NO: 186)CDR3: ALPRRGESELPSTVKEYGYW hzH10opt (SEQ ID NO: 77)EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWEGVGAYYAESVKGRFTISKDNAKRTLYLQMSSLRAEDTAVYYCALPRRGDAELPSTVKEYGYWGQGTLVTVKPGG (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 184) CDR2: AIRWEGVGAY (SEQ ID NO: 187)CDR3: ALPRRGDAELPSTVKEYGYW hzH10v10opt (SEQ ID NO: 78)EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDAVGAYYAESVKGRFTISKDNAKRTLYLQMSSLRAEDTAVYYCALPRRGESELPSTVKEYGYWGQGTLVTVKPGG (SEQ ID NO: 181) CDR1: SVSTFGTSPV(SEQ ID NO: 185) CDR2: AIRWDAVGAY (SEQ ID NO: 186)CDR3: ALPRRGESELPSTVKEYGYW H11 (SEQ ID NO: 79)QLQLQESGGGLVQAGDSLRLSCQVSGRTLSAYLMAWFRQAPNKVREYLGRIRWNEGDTYYPDSVKGRFTISKDDAKNTVYLRMNSLKPEDTAVYYCAARS IFNPSDQYVYWGQGTQVTVSS(SEQ ID NO: 188) CDR1: SGRTLSAYLM (SEQ ID NO: 189) CDR2: RIRWNEGDTY(SEQ ID NO: 190) CDR3: AARSIFNPSDQYVYW hzH11v1 (SEQ ID NO: 80)EVQLLESGGGLVQPGGSLRLSCAASGRTLSAYLMSWFRQAPGKGLEYVSAIRWNEGDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAARS IFNPSDQYVYWGQGTLVTVSS(SEQ ID NO: 188) CDR1: SGRTLSAYLM (SEQ ID NO: 28) CDR2: AIRWNEGDTY(SEQ ID NO: 190) CDR3: AARSIFNPSDQYVYW hzH11v2 (SEQ ID NO: 81)EVQLLESGGGLVQPGGSLRLSCAVSGRTLSAYLMSWFRQAPGKGREYVSRIRWNEGDTYYADSVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCAARS IFNPSDQYVYWGQGTLVTVSS(SEQ ID NO: 188) CDR1: SGRTLSAYLM (SEQ ID NO: 189) CDR2: RIRWNEGDTY(SEQ ID NO: 190) CDR3: AARSIFNPSDQYVYW 1F10 (SEQ ID NO: 82)EVQLVQSGGGLVQAGGSLRLSCAASGSTFSSLDMGWFRQAPGKERAFVAAISRSGDNIYYAESVKGRFTISRDNAENTMYLQMNSLKPEDSAVYYCAVESQPTYSGGVYYPRYGMDVWGQGTQVTVSS (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 147)CDR3: AVESQPTYSGGVYYPRYGMDVW hz1F10 (SEQ ID NO: 83)EVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMSWFRQAPGKGLEFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVESQPTYSGGVYYPRYGMDVWGQGTLVTVSS (SEQ ID NO: 31) CDR1: SGSTFSSLDMSW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 147)CDR3: AVESQPTYSGGVYYPRYGMDVW hz1F10v2 (SEQ ID NO: 84)EVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVESQPTYSGGVYYPRYGMDVWGQGTLVTVSS (SEQ ID NO: 142) CDR1: SGSTFSSLDMGW(SEQ ID NO: 143) CDR2: AISRSGDNIY (SEQ ID NO: 147)CDR3: AVESQPTYSGGVYYPRYGMDVW 2x_1F5-DS (SEQ ID NO: 92)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVCAIYWSGGTVYYAESVKGRFTCSRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVCAIYWSGGTVYYAESVKGRFTCSRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTV KPGGGG 2x_1F5(SEQ ID NO: 93) EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVSAIYWSGGTVYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVSAIYWSGGTVYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTV KPGGGG 2x_1F5_gs6(SEQ ID NO: 94) EVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGMDVWGQGTLVTVSGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGMD VWGQGTLVTVSSAGGGG2x_1F5_gs12 (SEQ ID NO: 95)EVQLLESGGGLVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVSAIYWSGGTVYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVSSGGGSGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVSAIYWSGGTVYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQ GTLVTVSSAGGGG2x_1F5_gs15 (SEQ ID NO: 96)EVQLLESGGGLVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVSAIYWSGGTVYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGLTFPNYGMSWFRQAPGKGLEFVSAIYWSGGTVYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVTIRGAATQTWKYDY WGQGTLVTVSSAGGGG2x_hz1F2v2-gs6 (SEQ ID NO: 97)EVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGMDVWGQGTLVTVSGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGMD VWGQGTLVTVSSAGGGG2x_hz1F2v2-gs9 (SEQ ID NO: 98)EVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGMDVWGQGTLVTVSSGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVDSQPTYSGGVYYPRY GMDVWGQGTLVTVSSAGGGG2x_hz1F2v2-gs12 (SEQ ID NO: 99)EVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGMDVWGQGTLVTVSSGGGSGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGMDVWGQGTLVTVSSAGGGG 2x_hz1F2v2-gs15 (SEQ ID NO: 100)EVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMSWFRQAPGKGLEFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGMDVWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGSTFSSLDMSWFRQAPGKGLEFVSAISRSGDNIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVDSQPTYSGGVYYPRYGMDVWGQGTLVTVSSAGGGG 2x_hzB04v1-gs6 (SEQ ID NO: 101)EVQLLESGGGLVQPGGSLRLSCAASGRAFSNYAMSWFRQAPGKGLEFVSAINWNGENRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALSFRLGGEPYGDAYWGQGTLVTVSGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRAFSNYAMSWFRQAPGKGLEFVSAINWNGENRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALSFRLGGEPYGDAYWGQGTLV TVSSAGGGG2x_hzB04v1-gs12 (SEQ ID NO: 102)EVQLLESGGGLVQPGGSLRLSCAASGRAFSNYAMSWFRQAPGKGLEFVSAINWNGENRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALSFRLGGEPYGDAYWGQGTLVTVSSGGGSGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRAFSNYAMSWFRQAPGKGLEFVSAINWNGENRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALSFRLGGEPYGDAYW GQGTLVTVSSAGGGG2x_hzB04v1-gs15 (SEQ ID NO: 103)EVQLLESGGGLVQPGGSLRLSCAASGRAFSNYAMSWFRQAPGKGLEFVSAINWNGENRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALSFRLGGEPYGDAYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRAFSNYAMSWFRQAPGKGLEFVSAINWNGENRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAALSFRLGGEPYGD AYWGQGTLVTVSSAGGGG2x_F03v2-gs6 (SEQ ID NO: 104)EVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMGWFRQAPGKEREFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVSGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMGWFRQAPGKEREFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGT LVTVSSAGGGG2x_F03v1-gs6 (SEQ ID NO: 105)EVQLLESGGGKVQPGGSLRLSCAASGRSISNYAMSWFRQAPGKGLEFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVSGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMSWFRQAPGKGLEFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGT LVTVSSAGGGG2x_F03v1-gs9 (SEQ ID NO: 106)EVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMSWFRQAPGKGLEFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVSSGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMSWFRQAPGKGLEFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWG QGTLVTVSSAGGGG2x_F03v1-gs12 (SEQ ID NO: 107)EVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMSWFRQAPGKGLEFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVSSGGGSGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMSWFRQAPGKGLEFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGAN YWGQGTLVTVSSAGGGG2x_F03v1-gs15 (SEQ ID NO: 108)EVQLLESGGGKVQPGGSLRLSCAASGRSISNYAMSWFRQAPGKGLEFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVSGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMSWFRQAPGKGLEFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGT LVTVSSAGGGG2x_hzH10v2-gs6 (SEQ ID NO: 109)EVQLLESGGGLVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVSGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQ GTLVTVSSAGGGG2x_hzH10v2-gs15 (SEQ ID NO: 110)EVQLLESGGGLVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVSSAGGGG 2x_hz1F5v3_gs6 (SEQ ID NO: 111)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFVSAIYWSGGTVFYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFVSAIYWSGGTVFYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTV KPGGGGDKTHTCPPC2x_hz1F5v4_gs6 (SEQ ID NO: 112)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFLAVIYWSGGTVFYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFLAVIYWSGGTVFYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTV KPGGGGDKTHTCPPC2x_hz1F5v5_gs6 (SEQ ID NO: 113)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFVSAIYWSGGTVYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFVSAIYWSGGTVYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTV KPGGGGDKTHTCPPC2x_hz1F5v6_gs6 (SEQ ID NO: 114)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFLAVIYWSGGTVYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFLAVIYWSGGTVYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTV KPGGGGDKTHTCPPC2x_hz1F5v7_gs6 (SEQ ID NO: 115)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFVSAIYWSGGTVYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFVSAIYWSGGTVYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTV KPGGGGDKTHTCPPC2x_hz1F5v8_gs6 (SEQ ID NO: 116)EVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFLAVIYWSGGTVYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGLTFPNYGMGWFRQAPGKEREFLAVIYWSGGTVYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAVTIRGAATQTWKYDYWGQGTLVTV KPGGGGDKTHTCPPC2x_hzC06v2_gs6 (SEQ ID NO: 117)EVQLLESGGGEVQPGGSLRLSCAASGRTVSNYAMGWFRQAPGKDREFVSALNWGGDTTYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAAAQSFRRGGAPYGDNYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGRTVSNYAMGWFRQAPGKDREFVSALNWGGDTTYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAAAQSFRRGGAPYGDNYWGQGTLV TVKPGGGGDKTHTCPPC2x_hzC06v2_gs9 (SEQ ID NO: 118)EVQLLESGGGEVQPGGSLRLSCAASGRTVSNYAMGWFRQAPGKDREFVSALNWGGDTTYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAAAQSFRRGGAPYGDNYWGQGTLVTVKPGGSGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGRTVSNYAMGWFRQAPGKDREFVSALNWGGDTTYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAAAQSFRRGGAPYGDNYWGQG TLVTVKPGGGGDKTHTCPPC3x_hzF03 (SEQ ID NO: 119)EVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMSWFRQAPGKGLEFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMSWFRQAPGKGLEFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRSISNYAMSWFRQAPGKGLEFVSASVWNNGGNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVVARTPETPITSARGANYWGQGTL VTVSSAGGGG 3x_H10-DS(SEQ ID NO: 120) EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVCAIRWEGVGAYYAESVKGRFTCSRDNAKNTLYLQMSSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVCAIRWEGVGAYYAESVKGRFTCSRDNAKNTLYLQMSSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVCAIRWEGVGAYYAESVKGRFTCSRDNAKNTLYLQMSSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVKPGGGG 3x_H10 (SEQ ID NO: 121)EVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWEGVGAYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWEGVGAYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWEGVGAYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTLVTVKPGGGG 3x_1F2-DS (SEQ ID NO: 122)EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVCAISRSGDNIYYAESVKGRFTCSRDNAKNTLYLQMSSLRAEDTAVYYCAVESQPTYSGGVYYPRYGMDVWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVCAISRSGDNIYYAESVKGRFTCSRDNAKNTLYLQMSSLRAEDTAVYYCAVESQPTYSGGVYYPRYGMDVWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVCAISRSGDNIYYAESVKGRFTCSRDNAKNTLYLQMSSLRAEDTAVYYCAVESQPTYSGGVYYPRYGMDVWGQGTLVTVKPGGGG 3x_1F2(SEQ ID NO: 123) EVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVESQPTYSGGVYYPRYGMDVWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVESQPTYSGGVYYPRYGMDVWGQGTLVTVKPGGSGGSEVQLLESGGGEVQPGGSLRLSCAASGSTFSSLDMGWFRQAPGKGREFVSAISRSGDNIYYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAVESQPTYSGGVYYPRYGMDVWGQGTLVTVKPGGGG 3x_H10-gs15(SEQ ID NO: 124) QVQLVQSGGGLVQAGGSLTLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYADSVRGRFKNSKDNAKRTAYLQMNRLKPEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVQSGGGLVQAGGSLTLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYADSVRGRFKNSKDNAKRTAYLQMNRLKPEDTAVYYCALPRRGDSELPSTVKEYGYWGQGTQVTVSSGGGGSGGGGSGGGGSQVQLVQSGGGLVQAGGSLTLSCAASVSTFGTSPVGWFRQAPGKEREFVSAIRWDGVGAYYADSVRGRFKNSKDNAKRTAYLQMNRLKPEDTAVYYCALPRRGDSELPSTVKEYGYWGQ GTQVTVSSAGGGGTAS266/11H6_hu_tetramer (SEQ ID NO: 125)EVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSS FIX-TAS266 (SEQ ID NO: 126)EVQLLESGGGEVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVKPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGEVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVKPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGEVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVKPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGEVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVKPGG

The DR5-targeting proteins described herein are useful in a variety oftherapeutic, diagnostic and prophylactic indications. For example, theDR5-targeting proteins are useful in treating a variety of diseases anddisorders in a subject. In some embodiments, the DR5-targeting proteinsare useful in treating, alleviating a symptom of, ameliorating and/ordelaying the progression of a disease or disorder in a subject sufferingfrom or identified as being at risk for an inflammatory disease ordisorder. In some embodiments, the DR5-targeting proteins are useful intreating, alleviating a symptom of, ameliorating and/or delaying theprogression of a cancer or other neoplastic condition. In someembodiments, the cancer is bladder cancer, breast cancer,uterine/cervical cancer, ovarian cancer, prostate cancer, testicularcancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer,colorectal cancer, colon cancer, kidney cancer, head and neck cancer,lung cancer, stomach cancer, germ cell cancer, bone cancer, livercancer, thyroid cancer, skin cancer, neoplasm of the central nervoussystem, lymphoma, leukemia, myeloma, sarcoma, mesothelioma, leukemia,lymphoma, myeloma, and virus-related cancer. In certain embodiments, thecancer is a metastatic cancer, refractory cancer, or recurrent cancer.In some embodiments, the DR5-targeting proteins are useful in reducingor depleting the number of T regulatory cells in a tumor of a subject inneed thereof. In some embodiments, the DR5-targeting proteins are usefulin stimulating an immune response in a subject. In some embodiments, theDR5-targeting proteins are useful in treating, alleviating a symptom of,ameliorating and/or delaying the progression of an autoimmune disease ordisorder. In some embodiments, the DR5-targeting proteins are useful intreating, alleviating a symptom of, ameliorating and/or delaying theprogression of viral, bacterial and parasitic infections.

Therapeutic formulations of the disclosure, which include aDR5-targeting molecule of the disclosure, are used to treat or alleviatea symptom associated with a disease or disorder associated with aberrantactivity and/or expression of DR5 in a subject. A therapeutic regimen iscarried out by identifying a subject, e.g., a human patient sufferingfrom (or at risk of developing) a disease or disorder associated withaberrant activity and/or expression of DR5 using standard methods,including any of a variety of clinical and/or laboratory procedures. Theterm patient includes human and veterinary subjects. The term subjectincludes humans and other mammals.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular disease or disorderassociated with aberrant activity and/or expression of DR5. Alleviationof one or more symptoms of the disease or disorder associated withaberrant activity and/or expression of DR5 indicates that theDR5-targeting molecule confers a clinical benefit.

Therapeutic uses of the DR5-targeting molecules of the disclosure canalso include the administration of one or more additional agents.

In some embodiments, the DR5-targeting molecule is administered duringand/or after treatment in combination with one or more additionalagents. In some embodiments, the DR5-targeting molecule and theadditional agent are formulated into a single therapeutic composition,and the DR5-targeting molecule and additional agent are administeredsimultaneously. Alternatively, the DR5-targeting molecule and additionalagent are separate from each other, e.g., each is formulated into aseparate therapeutic composition, and the DR5-targeting molecule and theadditional agent are administered simultaneously, or the DR5-targetingmolecule and the additional agent are administered at different timesduring a treatment regimen. For example, the DR5-targeting molecule isadministered prior to the administration of the additional agent, theDR5-targeting molecule is administered subsequent to the administrationof the additional agent, or the DR5-targeting molecule and theadditional agent are administered in an alternating fashion. Asdescribed herein, the DR5-targeting molecule and additional agent areadministered in single doses or in multiple doses.

In some embodiments, the DR5-targeting molecule and the additionalagent(s) are administered simultaneously. For example, the DR5-targetingmolecule and the additional agent(s) can be formulated in a singlecomposition or administered as two or more separate compositions. Insome embodiments, the DR5-targeting molecule and the additional agent(s)are administered sequentially, or the DR5-targeting molecule and theadditional agent are administered at different times during a treatmentregimen.

Methods for the screening of DR5 targeting molecules that possess thedesired specificity include, but are not limited to, enzyme linkedimmunosorbent assay (ELISA), enzymatic assays, flow cytometry, and otherimmunologically mediated techniques known within the art.

The disclosure further provides nucleic acid sequences and particularlyDNA sequences that encode the present fusion proteins. Preferably, theDNA sequence is carried by a vector suited for extrachromosomalreplication such as a phage, virus, plasmid, phagemid, cosmid, YAC, orepisome. In particular, a DNA vector that encodes a desired fusionprotein can be used to facilitate the methods of preparing theDR5-targeting molecules described herein and to obtain significantquantities of the fusion protein. The DNA sequence can be inserted intoan appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. A variety of host-vector systems may beutilized to express the protein-coding sequence. These include mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Dependingon the host-vector system utilized, any one of a number of suitabletranscription and translation elements may be used.

The disclosure also provides methods of producing a DR5-targetingmolecule by culturing a cell under conditions that lead to expression ofthe polypeptide, wherein the cell comprises an isolated nucleic acidmolecule encoding a DR5-targeting molecule described herein, and/orvectors that include these isolated nucleic acid sequences. Thedisclosure provides methods of producing a DR5-targeting molecule byculturing a cell under conditions that lead to expression of theDR5-targeting molecule, wherein the cell comprises an isolated nucleicacid molecule encoding a DR5-targeting molecule described herein, and/orvectors that include these isolated nucleic acid sequences.

The fusion proteins of the disclosure (also referred to herein as“active compounds”), and derivatives, fragments, analogs and homologsthereof, can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the fusionprotein and a pharmaceutically acceptable carrier. As used herein, theterm “pharmaceutically acceptable carrier” is intended to include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensionscan also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a kit, container,pack, or dispenser together with instructions for administration. Thesepharmaceutical compositions can be included in diagnostic kits withinstructions for use.

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients. The term patientincludes human and veterinary subjects.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the terms “targeting fusion protein” and “antibody” canbe synonyms. As used herein, the term “antibody” refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin (Ig) molecules, i.e., molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen. By“specifically bind” or “immunoreacts with” “or directed against” ismeant that the antibody reacts with one or more antigenic determinantsof the desired antigen and does not react with other polypeptides orbinds at much lower affinity (K_(d)>10⁻⁶). Antibodies include, but arenot limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody),single chain, Fab, Fab, and F(ab′)₂ fragments, F_(v), scFvs, an Fabexpression library, and single domain antibody (sdAb) fragments, forexample V_(H)H, V_(NAR), engineered V_(H) or V_(K).

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses (also known as isotypes) as well, such as IgG₁, IgG₂, andothers. Furthermore, in humans, the light chain may be a kappa chain ora lambda chain.

The term “monoclonal antibody” (mAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three-dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

The single domain antibody (sdAb) fragments portions of the fusionproteins of the present disclosure are referred to interchangeablyherein as targeting polypeptides herein.

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to/by an immunoglobulin or fragment thereof,or a T-cell receptor. The term “epitope” includes any proteindeterminant capable of specific binding to/by an immunoglobulin orT-cell receptor. Epitopic determinants usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics. An antibodyis said to specifically bind an antigen when the dissociation constantis ≤1 μM; e.g., ≤100 nM, preferably ≤10 nM and more preferably ≤1 nM.

As used herein, the terms “immunological binding” and “immunologicalbinding properties” and “specific binding” refer to the non-covalentinteractions of the type which occur between an immunoglobulin moleculeand an antigen for which the immunoglobulin is specific. The strength,or affinity of immunological binding interactions can be expressed interms of the dissociation constant (K_(d)) of the interaction, wherein asmaller K_(d) represents a greater affinity. Immunological bindingproperties of selected polypeptides can be quantified using methods wellknown in the art. One such method entails measuring the rates ofantigen-binding site/antigen complex formation and dissociation, whereinthose rates depend on the concentrations of the complex partners, theaffinity of the interaction, and geometric parameters that equallyinfluence the rate in both directions. Thus, both the “on rate constant”(k_(on)) and the “off rate constant” (k_(off)) can be determined bycalculation of the concentrations and the actual rates of associationand dissociation. (See Nature 361:186-87 (1993)). The ratio ofk_(off)/k_(on) enables the cancellation of all parameters not related toaffinity, and is equal to the dissociation constant K_(d). (See,generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). Anantibody of the present disclosure is said to specifically bind to anantigen, when the equilibrium binding constant (K_(d)) is ≤1 μM,preferably ≤100 nM, more preferably ≤10 nM, and most preferably ≤100 pMto about 1 pM, as measured by assays such as radioligand binding assays,surface plasmon resonance (SPR), flow cytometry binding assay, orsimilar assays known to those skilled in the art.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present disclosure, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with thedisclosure.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has specific bindingto DR5, under suitable binding conditions. Typically, polypeptideanalogs comprise a conservative amino acid substitution (or addition ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, preferably at least 50 aminoacids long or longer, and can often be as long as a full-lengthnaturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986), Veber and Freidinger TINS p.392 (1985); and Evans et al. J. Med.Chem. 30:1229 (1987). Such compounds are often developed with the aid ofcomputerized molecular modeling. Peptide mimetics that are structurallysimilar to therapeutically useful peptides may be used to produce anequivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, CH(OH)CH₂—,and —CH₂SO—, by methods well known in the art. Systematic substitutionof one or more amino acids of a consensus sequence with a D-amino acidof the same type (e.g., D-lysine in place of L-lysine) may be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, and/or an extractmade from biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing and/or ameliorating a disorder and/or symptomsassociated therewith. By “alleviate” and/or “alleviating” is meantdecrease, suppress, attenuate, diminish, arrest, and/or stabilize thedevelopment or progression of a disease such as, for example, a cancer.It will be appreciated that, although not precluded, treating a disorderor condition does not require that the disorder, condition or symptomsassociated therewith be completely eliminated.

In this disclosure, “comprises,” “comprising,” “containing,” “having,”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; the terms“consisting essentially of” or “consists essentially” likewise have themeaning ascribed in U.S. Patent law and these terms are open-ended,allowing for the presence of more than that which is recited so long asbasic or novel characteristics of that which is recited are not changedby the presence of more than that which is recited, but excludes priorart embodiments.

By “effective amount” is meant the amount required to ameliorate thesymptoms of a disease relative to an untreated patient. The effectiveamount of active compound(s) used to practice the present disclosure fortherapeutic treatment of a disease varies depending upon the manner ofadministration, the age, body weight, and general health of the subject.Ultimately, the attending physician or veterinarian will decide theappropriate amount and dosage regimen. Such amount is referred to as an“effective” amount.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, rodent, ovine,primate, camelid, or feline.

The term “administering,” as used herein, refers to any mode oftransferring, delivering, introducing, or transporting a therapeuticagent to a subject in need of treatment with such an agent. Such modesinclude, but are not limited to, oral, topical, intravenous,intraperitoneal, intramuscular, intradermal, intranasal, andsubcutaneous administration.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, theterms “a,” “an,” and “the” are understood to be singular or plural.Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromthe context, all numerical values provided herein are modified by theterm “about.”

The invention will be further described in the following examples, whichdo not limit the scope of the disclosure described in the claims.

EXAMPLES Example 1: Binding Assays

Binding of DR5-targeting fusion proteins was assessed by flow cytometry,using a CHO cell line stably transfected with cDNA encoding full lengthDR5 or cancer cell lines that endogenously express DR5. A titrationseries of the fusion protein was incubated with the DR5-expressing celllines (approx. 2.5-5×10⁴ cells/well) for 30 minutes at 4° C. in FACSBuffer (PBS 1% BSA, 0.1% NaN₃ pH 7.4) in 96 well plates. Following 3wash steps in FACS buffer, an APC-conjugated anti-human Fcγ specificsecondary antibody (Jackson ImmunoResearch) was added and incubated for30 minutes at 4° C. Following three additional wash steps in FACS bufferbound antibody was detected via flow cytometry (IQue Intellicyte).Binding of fusion proteins to cynomologus monkey DR5 (cynoDR5) wasdetermined by ELISA wherein a recombinant protein corresponding to theextracellular domain (ECD) of cynoDR5 fused to a murine Fc region (mFc)was immobilized on Medisorp 96 well plates (Nunc). Following sufficientblocking and washing steps, bound fusion proteins were detected using anHRP-conjugated anti-human Fcγ specific secondary antibody (JacksonImmunoResearch) and TMB reagent and absorbance read at A_(650nm).

Example 2: Apoptosis Assays

Antibody-mediated direct killing of cells was determined by measuringthe amount of ATP present following a 16-48 h treatment period usingCellTiter-Glo® (Promega G7572). Cancer cells were seeded at 1.5-3×10⁴cells/well at 7×10⁴ cells/well in 96-well flat-bottom tissue culturetreated plates. An alternative method for measuring cell death is tofluorescently stain cells using IncuCyte™ Caspase-3/7 Reagent forApoptosis (Essen BioScience 4440) during antibody treatment and quantifyfluorescent cells using an IncuCyte® ZOOM System some embodiments, thefusion protein contains a polypeptide. Cell lines used include Colo-205(ATCC® CCL-222™), Panc-1 (ATCC® CRL-1469™), HCT-116 (ATCC® CCL-247™),JL-1 (DSMZ ACC 596), NCI-H28 (ATCC® CRL-5820™), NCI-H460 (ATCC®HTB-177™), HT-29 (ATCC® HTB-38™). MSTO-211H (ATCC® CRL-2081™). In someexperiments, an anti-human IgG Fcγ-specific secondary (JacksonImmunoResearch) antibody was used to crosslink and further cluster theDR5 targeting fusion proteins of the present disclosure. In otherexperiments 6 μM doxycycline was used to sensitize cells to DR5-mediatedapoptosis.

Example 3: Pre-Existing Autoantibodies Recognizing sdAbs

Pre-existing human anti-VH (HAVH) in human plasma or IVIG (purified IgGfrom pooled human plasma, trade name Gamunex®-C) were measured by ELISA.Test articles (TAS266, fusion proteins or therapeutic antibodies) werecoated on an ELISA plate in PBS, the plate was blocked by 3% BSA in PBS,then human plasma or IVIG (as a source of naturally occurring HAVH) wasdiluted in PBS+0.1% polysorbate-20 (PBST) and allowed to bind to theplate. After washing the plate with PBST, bound plasma antibodies (HAVH)were detected by anti-light chain secondary antibodies (anti-humanIgKappa or anti-IgLambda) conjugated to HRP, and developed with TMBsubstrate. This strategy of detecting HAVH by anti-light chain secondaryantibody is compatible with test articles lacking light chains, whichincludes TAS266 as well as the described multivalent sdAbs, andfacilitates detection of HAVH of any isotype. Control therapeuticantibodies with kappa or lambda light chains were coated and used as100% binding reference data points to normalize the data to, and servedas control IgG for the opposite secondary antibody.

Example 4: Hepatotoxicity Assays

Primary human hepatocytes or HepRG™ (Thermo Fisher Scientific) theterminally differentiated hepatic cells derived from a hepaticprogenitor cell line were used to assess DR5 agonist mediated apoptosisof hepatocytes. All assays were conducted in a similar manner to theapoptosis assays using cancer cell lines (Example 2). Pooled human IgGfrom multiple donors, IVIG (Gamunex®-C, Grifols), was used as source ofnatural sdAb-directed autoantibodies, also termed human anti-VH (HAVH)autoantibodies. In some experiments, IVIG was titrated or used at afixed concentration. In some assays, FIX-TAS266, which is a modifiedversion of TAS266 that is engineered to avoid recognition by HAVHautoantibodies, was included. FIX-2TAS66 includes modifications a Leu11and the C-terminal region of each of the four DR5 sdAbs of TAS266.

What is claimed is:
 1. An isolated polypeptide that binds death receptor5 (DR5) and comprises a plurality of DR5 binding domains (DR5BDs),wherein each DR5BD is a VHH comprising a CDR1 comprising the amino acidsequence of SEQ ID NO: 128, a CDR2 comprising the amino acid sequence ofSEQ ID NO: 131, and a CDR3 comprising the amino acid sequence of SEQ IDNO: 130, and wherein adjacent DR5BDs are operably linked by an aminoacid linker.
 2. The isolated polypeptide of claim 1, wherein theplurality of DR5BDs is two DR5BDs.
 3. The isolated polypeptide of claim1, wherein the plurality of DR5BDs is four DR5BDs.
 4. The isolatedpolypeptide of claim 1, wherein the plurality of DR5BDs is six DR5BDs.5. The isolated polypeptide of claim 1, wherein each DR5BD comprises theamino acid sequence of SEQ ID NO:
 87. 6. The isolated polypeptide ofclaim 2, wherein each DR5BD comprises the amino acid sequence of SEQ IDNO:
 87. 7. The isolated polypeptide of claim 3, wherein each DR5BDcomprises the amino acid sequence of SEQ ID NO:
 87. 8. The isolatedpolypeptide of claim 4, wherein each DR5BD comprises the amino acidsequence of SEQ ID NO:
 87. 9. The isolated polypeptide of claim 3,wherein the polypeptide is a homodimer of the structure:DR5BD-Linker-DR5BD-Linker-Hinge-Fc, where each DR5BD is a humanized VHHsequence.
 10. The isolated polypeptide of claim 9, wherein each DR5BDcomprises the amino acid sequence of SEQ ID NO:
 87. 11. The isolatedpolypeptide of claim 1, wherein the isolated polypeptide comprises animmunoglobulin hinge region and an immunoglobulin Fc region.
 12. Theisolated polypeptide of claim 11, wherein the immunoglobulin hingeregion comprises an amino acid sequence selected from EPKSSDKTHTCPPC(SEQ ID NO: 6), DKTHTCPPC (SEQ ID NO: 7), ESKYGPPCPPC (SEQ ID NO: 8).13. The isolated polypeptide of claim 11, wherein the immunoglobulin Fcregion is an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, or anIgG1 Fc region.
 14. The isolated polypeptide of claim 11, wherein theimmunoglobulin Fc region comprises an amino acid sequence selected fromSEQ ID NOs: 1-5 or
 127. 15. The isolated polypeptide of claim 1, whereinthe polypeptide comprises the amino acid sequence of SEQ ID NO:
 113. 16.The isolated polypeptide of claim 15, wherein the polypeptide is ahomodimer of the amino acid sequence of SEQ ID NO: 113 fused to an Fcregion polypeptide.
 17. The isolated polypeptide of claim 1, whereineach amino acid linker consists of 5-20 amino acids.
 18. The isolatedpolypeptide of claim 17, wherein each amino acid linker is composedpredominantly of glycine and serine.
 19. The isolated polypeptide ofclaim 18, wherein each amino acid linker comprises an amino acidsequence selected from GGSGGS (SEQ ID NO: 11); GGSGGSGGS (SEQ ID NO:12); GGSGGSGGSGGS (SEQ ID NO: 13); and GGSGGSGGSGGSGGS (SEQ ID NO: 14).20. The isolated polypeptide of claim 10, wherein each amino acid linkerconsists of 5-20 amino acids.
 21. The isolated polypeptide of claim 20,wherein each amino acid linker is composed predominantly of glycine andserine.
 22. The isolated polypeptide of claim 21, wherein each aminoacid linker comprises an amino acid sequence selected from GGSGGS (SEQID NO: 11); GGSGGSGGS (SEQ ID NO: 12); GGSGGSGGSGGS (SEQ ID NO: 13); andGGSGGSGGSGGSGGS (SEQ ID NO: 14).
 23. The isolated polypeptide of claim1, wherein each VHH is a humanized VHH.
 24. An isolated polypeptide thatbinds death receptor 5 (DR5), wherein the polypeptide is a homodimer ofthe amino acid sequence of SEQ ID NO: 113 fused to an Fc regionpolypeptide of SEQ ID NO: 2.